Regulatory t cell epitopes, compositions and uses thereof

ABSTRACT

The invention is directed to T cell epitopes wherein said epitopes comprises a peptide or polypeptide chain comprising at least a portion of an immunoglobulin constant or variable region. The invention also relates to methods of using and methods of making the epitopes of the invention.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/530,104, filed Aug. 2, 2019, which is acontinuation application of U.S. patent application Ser. No. 16/015,784,filed on Jun. 22, 2018, which is a continuation application of U.S.patent application Ser. No. 14/857,693, filed on Sep. 17, 2015, now U.S.Pat. No. 10,213,496, which is a continuation application of U.S. patentapplication Ser. No. 12/981,098, filed Dec. 29, 2010, which a divisionalapplication of U.S. patent application Ser. No. 12/021,832, filed onJan. 29, 2008, which claims priority to U.S. Provisional PatentApplication Ser. No. 60/898,347, filed Jan. 30, 2007. The entirecontents of all of the above-listed applications are incorporated byreference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 20, 2018, isnamed SEQENCE LISTING_ST25.txt and is 68 KB is size.

FIELD OF THE INVENTION

The invention relates generally to a novel class of T cell epitopecompositions (termed “Tregitopes”). The invention provides Tregitopecompositions, methods for their preparation and use.

BACKGROUND

Artificial induction of tolerance to self or to foreign antigens is thegoal of therapy for autoimmunity, transplantation allergy and otherdiseases, and is also desirable in the context of therapy withautologous proteins and non-autologous proteins. Until recently,therapeutic tolerance induction relied on broad-based approaches thatresulted in cellular depletion and cytokine profile alteration. Thesebroad-based approaches weaken the immune system in general and leavemany subjects vulnerable to opportunistic infections, autoimmune attackand cancer. There is a need in the art for less aggressive and moretargeted approaches to the induction of immune tolerance.

Immune tolerance is regulated by a complex interplay between T cells, Bcells, cytokines and surface receptors. Initial self/non-selfdiscrimination occurs in the thymus during neonatal development wheremedullary epithelial cells express specific self protein epitopes toimmature T cells. T cells recognizing self antigens with high affinityare deleted, but autoreactive T cells with moderate affinity sometimesavoid deletion and can be converted to so called ‘natural’ regulatory Tcells (T_(Reg)) cells. These natural T_(Reg) cells are exported to theperiphery and provide for constant suppression of autoimmunity.

A second form of tolerance occurs in the periphery where mature T cellsare converted to an ‘adaptive’ T_(Reg) phenotype upon activation viatheir T cell receptor in the presence of IL-10 and TGF-β. The possibleroles for these ‘adaptive’ T_(Reg) cells include dampening immuneresponse following the successful clearance of an invading pathogen as ameans of controlling excessive inflammation as might be caused by anallergic reaction or low level chronic infection, or possibly tofacilitate co-existence with beneficial symbiotic bacteria and viruses.‘Adaptive’ T_(Reg) may also play a role in managing the life cycle ofhuman antibodies that have undergone somatic hypermutation.

Natural regulatory T cells are a critical component of immune regulationin the periphery. Upon activation through their TCR natural Tregs arecapable of suppressing bystander effector T cell responses to unrelatedantigens through contact dependent and independent mechanisms. Inaddition the cytokines released by these cells including IL-10 andTGF-β, are capable of inducing antigen-specific adaptive Tregs. Despiteextensive efforts, with few exceptions, the antigen specificity ofnatural Tregs, and more importantly natural Tregs circulating inclinically significant volumes, is still unknown.

There is need in the art for the identification of regulatory T cellepitopes contained in common autologous proteins such as IgG(“Tregitopes”) and for methods for related to their preparation and ofuse.

SUMMARY

The present invention harnesses the functions of regulatory T cells(T_(Reg)), particularly those cells that already regulate immuneresponses to foreign and self proteins in the periphery (pre-existing ornatural T_(Reg)). In one aspect, the invention provides T-cell epitopepolypeptide compositions.

The selective engagement and activation of pre-existing natural Tregthrough the use of Tregitopes and Tregitope-antigen fusions, istherapeutically valuable as a means of treatment for any disease orcondition marked by the presence of an unwanted immune response.Examples include the following: Autoimmune disease such as type 1diabetes, MS, Lupus, and RA; Transplant related disorders such as Graftvs. Host disease (GVHD); Allergic reactions; Immune rejection ofbiologic medicines such as monoclonal antibodies, replacement proteinssuch as FVIII or Insulin, the use of therapeutic toxins such asBotulinum toxin; and the management of immune response to infectiousdisease whether acute or chronic.

In one embodiment, the present invention is directed to a T-cell epitopepolypeptide composition comprising at least one polypeptide selectedfrom the group consisting of: SEQ ID NOS:4-58. In a particularembodiment, the invention is directed to a pharmaceutical compositioncomprising a polypeptide of the invention and a pharmaceuticallyacceptable carrier.

In one embodiment, the present invention is directed to a nucleic acidencoding at least one T-cell epitope polypeptide selected from the groupconsisting of: SEQ ID NOS:4-58. In a particular embodiment, theinvention is directed to a vector comprising a nucleic acid of theinvention. In another embodiment, the invention is directed to a cellcomprising a vector of the invention.

In one embodiment, the invention is directed to a method of treating orpreventing a medical condition in a subject in need thereof comprisingadministering a therapeutically effective amount of a T-cell epitopepolypeptide selected from the group consisting of: SEQ ID NOS:4-58. In aparticular embodiment, the medical condition is selected from the groupconsisting of: an allergy, an autoimmune disease, a transplant relateddisorder, graft versus host disease, an enzyme or protein deficiencydisorder, a hemostatic disorder, cancer, infertility; and a viral,bacterial or parasitic infection.

In one embodiment, the invention is directed to a kit for preventing ortreating a medical condition in a subject, wherein the kit comprises atleast one T-cell epitope polypeptide selected from the group consistingof: SEQ ID NOS:4-58.

In one embodiment, the present invention is directed to a method forexpanding a population of regulatory T cells, comprising: (a) providinga biological sample from a subject; and (b) isolating regulatory T-cellsfrom the biological sample; and contacting the isolated regulatoryT-cells with an effective amount of a Tregitope composition of theinvention under conditions wherein the T-regulatory cells increase innumber to yield an expanded regulatory T-cell composition, therebyexpanding the regulatory T-cells in the biological sample.

In one embodiment, the present invention is directed to a method forstimulating regulatory T cells in a biological sample, comprising: (a)providing a biological sample from a subject; (b) isolating regulatoryT-cells from the biological sample; and contacting the isolatedregulatory T-cells with an effective amount of a Tregitope compositionof the invention under conditions wherein the T-regulatory cells arestimulated to alter one or more biological function, thereby stimulatingthe regulatory T-cells in the biological sample.

In one embodiment, the present invention is directed to a method forrepressing immune response in a subject, comprising administering acomposition comprising a therapeutically effective amount of a peptidecomprising a Tregitope to the subject, wherein the peptide represses theimmune response. In a particular embodiment, the peptide suppresseseffector T cell response. In a particular embodiment, the peptidesuppresses helper T cell response. In another embodiment, the peptidesuppresses B cell response.

In one embodiment, the present invention is directed to a method ofsuppressing antigen specific immune response in a subject through theadministration of a therapeutically effective amount of a compositioncomprising one or more Tregitopes, wherein the one or more Tregitopesare either covalently bound, non-covalently bound or in admixture with aspecific target antigen resulting in the diminution of immune responseagainst the target antigen. In a particular embodiment, the suppressiveeffect is mediated by natural Treg. In another embodiment, thesuppressive effect is mediated by adaptive Treg. In another embodiment,the peptide suppresses effector T cell response. In another embodiment,the peptide suppresses helper T cell response. In another embodiment,the peptide suppresses B cell response. In a particular embodiment, thepeptide comprises a sequence selected from the group consisting of: SEQID NOS:4-58.

In one embodiment, the present invention is directed to a method forenhancing the immunogenicity of a vaccine delivery vector, comprisingidentification and removal of regulatory T cell epitopes. In aparticular embodiment, the T cell epitopes are selected from the groupconsisting of: SEQ ID NOS:4-58.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic diagram of immunoglobulin G (IgG).

FIG. 2 is a series of graphical representations comparing capped anduncapped peptides. PBMC were stimulated in culture for 8 days witheither CEF (positive control peptide pool) alone (top panels),CEF+Tregitope 289-amide (middle panels), or CEF+Tregitope-289-uncapped(bottom panels). As compared to incubation with CEF alone, co-incubationof CEF with Tregitope-289-amide 1) resulted in a higher percentage ofcells that had a regulatory phenotype (left panel) and 2) resulted in anassociated significant (p<0.0005) decrease in the interferon gammasecretion in response to CEF restimulation (right panel). By contrast,co-incubation with Tregitope 289-uncapped (bottom panels) resulted in nosignificant difference relative to incubation with CEF alone (toppanels).

FIG. 3 shows activation of natural Tregs in the presence of Tregitope.Human PBMCs were stimulated directly in vitro for four days in thepresence of tetanus toxin peptide (TT₈₃₀₋₈₄₄), Tregitope or no stimulus.Cells were stained extracellularly with anti-CD4 and anti-CD25 and thenintracellularly with FoxP3, and analyzed by flow cytometry. Incubationwith Tregitope increased the percentage of CD4+CD25+Foxp3+ T cells(53.6% of 644 cells) as compared to TT₈₃₀₋₈₄₄ (12.5% of 745 cells) or nostimulus (19.5% of 497 cells).

FIG. 4 is a series of bar graphs showing Tregitope induces anup-regulation of T-regulatory cytokines and chemokines anddown-regulation of T-effector cytokines and chemokines. Responses to C3drestimulation following initial stimulation with a) a pool ofimmunogenic peptides derived from C3d protein (black bars); b) C3dpeptides+Tregitope-167 (light grey bars) or c) C3d peptides+Tregitope134 (medium grey bars). Responses are shown as fold increase overbackground, which was no stimulus (control) in the secondary incubation.The respective baseline (background) values in pg/ml are indicatedwithin the x-axis labels. There was no significant difference in levelsof IL-4, TNFα or TGFβ1

FIG. 5 is a series of bar graphs showing responses to C3d peptiderestimulation following initial stimulation with a) a pool ofimmunogenic peptides derived from C3d protein (black bars) or b) C3dpeptides+Tregitope-289 (light grey bars). Responses are shown as foldincrease over background, which was no stimulus (control) in thesecondary incubation. For each cytokine, baseline (no re-stimulus,background) values are shown within the x-axis labels.

FIG. 6 is a bar graph showing co-stimulation with epitopes derived fromTSHR, the target antigen of Graves' disease, suppresses immune responseto the epitopes in PBMC from a patient with Graves' disease. PBMC from apatient with Graves' disease were first incubated for 8 days witheither 1) a pool of TSHR peptides alone or 2) a pool of TSHR peptidesand a pool of Tregitope-134, Tregitope-167, and Tregitope-289. Cellswere then harvested, washed and incubated (in IL-4 ELISpot plates asdescribed) with 1) individual TSHR peptides and the Tregitope pool or 2)the pool of TSHR peptides and the Tregitope pool. A “no restimulus”control was also plated. Responses are shown relative to restimulationwith no antigen. Black bars correspond to incubations and restimulationsdone with antigen alone, grey bars correspond to incubations andrestimulations with antigen+the Tregitope pool. In this experiment theTregitope co-incubation suppressed response to individual TSHR peptidesby 35% to 67% and suppressed the response to the pool of TSHR peptidesby 65%. P values for pairwise comparisons are shown.

FIG. 7 is a bar graph showing average response of three subjects inresponse to a commercially available pool of positive control peptides(CEF) following an eight day incubation with one of the following: CEFalone, CEF+Tregitope-289, CEF+Tregitope 294, CEF+Tregitope-029,CEF+Tregitope-074, or CEF+Tregitope-009. Responses to CEF are suppressedby 29 to 48% depending on the individual Tregitope used. Baselineresponses to CEF (in the samples previously incubated to CEF alone)ranged from 1404 to 10139 IFN-γ SFC/million PBMC over background(background is no restimulus). P values for pairwise comparisons areshown.

FIG. 8 is a bar graph showing co-incubation with Tregitope suppressesimmune response to peptide epitopes derived from Botulinum neurotoxin.Peripheral blood was drawn from a subject with evidence of anti-BoNT/Aantibodies. PBMC were first incubated for eight days with either a poolof BoNT/A peptides alone or pool of BoNT/A peptides+a pool of Tregitopes(Tregitope-134, Tregitope-167, and Tregitope-289). Cells were thenharvested, washed and then incubated (in IFN-γ ELISpot plates) withBoNT/A peptides individually in triplicate and in a pool in triplicate.Responses are shown relative to re-stimulation with no antigen. Blackbars correspond to incubations with antigen alone, grey bars correspondto incubations with antigen+the pool of Tregitopes. The response toBoNT/A was suppressed by 26% to 73%; the response to the pool of BoNT/Apeptides was suppressed by 59%. P values for pairwise comparisons areshown.

FIG. 9 is a series of graphs showing Tregitope-289 and Tregitope-134down-regulate proliferation in response to co-administeredimmunodominant antigens in vitro. PBMCs were isolated from a blood bankdonor and stimulated for eight days with CEF alone, CEF+Tregitope-134 orCEF+Tregitope-289. Co-incubation with either of the two Tregitopes leadto a decrease in response to CEF as measured by IFN-γ ELISA (left panel)and to a decrease in proliferation as measured by CFSE Fluorescence(right panel) (grey shaded refers to the suppressed responses withantigen-Tregitope co-incubation; the black refers to baseline responsesfollowing incubation with antigen alone).

FIG. 10 is a plot showing response to vaccinia peptide. PBMCs wereisolated from a blood bank donor and stimulated for eight days with CEFalone, CEF+Tregitope-134 or CEF+Tregitope-289. Co-incubation with eitherof the two Tregitopes led to a decrease in response to CEF as measuredby IFN-γ ELISA (left panel) and to a decrease in proliferation asmeasured by CFSE Fluorescence (right panel). Grey shaded refers to thesuppressed responses with antigen-Tregitope co-incubation; the blackrefers to baseline responses following incubation with antigen alone.

FIG. 11 is a series of graphs showing Tregitope suppression is mediatedby cells with a regulatory phenotype. Tregitope suppression is dependenton CD4+CD25Hi T cells. Left panel: PBMC from allergic individuals werestained with anti-CD4 and anti-CD25 antibodies and analyzed by flowcytometry. The CD4+CD25Hi subset (gate) was depleted from the remainingPBMC. Center panel: CD4+CD25Hi depleted and non-depleted PBMC wereco-stimulated HDM lysate with or without Tregitope-289. CD4+CD25Hidepleted PBMC were less able to suppress IFN-γ than non-depleted PBMC.Right panel: co-incubation of HDM lysate and Tregitope-289 leads todecreased proliferation of CD4+ cells in response to HDM lysatestimulus.

FIG. 12 is a plot and graph showing expansion of TReg (CD4/CD25 Hi)correlates with IL-10 secretion. The expansion of CD4 CD25hi T cellsfollowing co-incubation with Tregitope-289 and HDM; and the amount ofIL-10 secreted by the co-incubated cells following restimulation withHDM alone. Results for co-incubation with Tregitope-167 are similar:increase from 1.67% to 7.5% CD4/CD25hi cells and a five-fold increase inIL-10 secretion.

FIG. 13 is a series of graphs and plots showing Tregitope co-incubationcauses suppression of antigen-specific allergic Th2 response. Co-culturewith Tregitope and Bet v 1141-155 allergen causes a shift from Th2effector to Th1/T_(Reg). PBMC from three birch-tree-pollen-allergicsubjects were co-stimulated with Bet v 1141-155 peptide with or withoutTregitope-167. Ten day Tregitope co-stimulation led to a decrease inIL-5 secretion (lower left panel) and to a decrease in Th2-associatedsurface markers (top panel) by Bet v 1144-155 tetramer positive CD4+cells. Prolonged culture (30 days, lower right panel) led to asignificant shift from Th2 (IL-5) to Th1 (IFN-γ) in Bet v1144-155-specific cells.

FIG. 14 is a graph showing Tregitope co-administration causessuppression of effector responses to co-administered protein therapeuticin vivo. ANTIGEN-XX (see Example 5A) immunization alone (black bars)provokes a robust response by both IL-4 (left bars in pair, left axis)and anti-ANTIGEN-XX antibody titers (right bars in pair, right axis).These responses are both halved (grey bars) when ANTIGEN-XX isco-administered with the murine homologues of Tregitope-167 andTregitope-106. Responses to ANTIGEN-XX in sham-immunized animals arenegative as expected. Responses by Antibody (right bars in pair) andIL-4 ELISpot (left bars in pair) are correlated.

FIG. 15 is a graph showing IL-4 and antibody responses to house mitelysate (HDML) and dust mite antigen. HDML immunization (black bars)provokes a robust response by both IL-4 (Left bars in pair, left axis)and anti HDM Antigen antibody titers (Right bars in each pair, rightaxis). These responses are both halved (grey bars) when HDML isco-administered with the murine homologue of Tregitope-289. Responses toHDML in sham-immunized animals are negative as expected. Responses byAntibody (right bars in pair) and IL-4 ELISpot (left bars in pair) arecorrelated. These graphs show 38% suppression in vivo in DM naïve miceand 84% percent in the case of pre-sensitized mice. See Example 5B.

FIG. 16 is a bar graph showing in vivo suppression of T effectorresponse to immunogenic peptide therapeutic (“IPT”) by Tregitopeco-administration. HLA DR4 Tg mice were dosed three times subcutaneouslywith IPT alone or IPT+murine FC, or IPT+Tregitope-289 (murinehomologue). One week following the last dose, the mice were sacrificedand splenocytes were stimulated with the immunogenic peptide therapeuticin a 48 hr IL-4 ELISpot assy. Co-administration of immunogenic proteintherapeutic with either Fc or Tregitope-289 lead to a significantreduction in IL-4 spot-forming cells. See Example 5C.

FIG. 17 depicts an example of an immunogenic influenza HA peptide thatcontains an EpiBar and the EpiMartix analysis of the promiscuousinfluenza epitope. The influenza HA peptide scores extremely high forall eight alleles in EpiMatrix and has a cluster score of 18. Clusterscores of 10 are considered significant. The band-like EpiBar pattern ischaracteristic of promiscuous epitopes. Results are shown for PRYVKQNTL(SEQ ID NO:59), RYVKQNTLK (SEQ ID NO:60), YVKQNTLKL (SEQ ID NO:61),VKQNTLKLA (SEQ ID NO:62) and KQNTLKLAT (SEQ ID NO:63). Z score indicatesthe potential of a 9-mer frame to bind to a given HLA allele. All scoresin the top 5% are considered “hits”, while non hits (*) beow 10% aremasked in FIG. 17 for simplicity.

FIG. 18A and FIG. 18B depict the Tregitopes on the instant-disclosureand their EpiMatrix Scores.

FIG. 19 illustrates an exemplary BLAST report of Tregitope-289 (SEQ IDNO: 4) showing homology of Tregitope-289 to other organisms. Homologyanalysis of the IgG-derived Tregitopes to non-human species wasperformed by uploading the sequences into the Basic Local AlignmentSearch Tool (BLAST) via the NCBI website (ncbi.nlm.nih.gov/blast). TheBLAST program compares protein sequences to sequence databases andcalculates the statistical significance of matches in order to findregions of local similarity between sequences. The IgG-derivedTregitopes were found to be conserved across non-human species such asmouse, rat, cat, camel, cow and non-human primates.

FIG. 20 illustrates the binding of affinity of the Tregitopes of theinstant disclosure to each of 4 common HLA, showing that the instantTregitopes indentified by in silico analysis bound to human MHCmolecules. Soluble MHC binding assays were performed on the syntheticIgG Tregitopes of the instant disclosure according to the methodsdescribed below in the EXEMPLIFICATION section. ICso values (μM) werederived by a six point inhibition curve of a strong binding controlpeptide.

FIG. 21 depicts the results of interferon-γ ELISpot responses toBotulinum Toxin Antigen stimulus following incubation with or withoutTregitopes (spot forming cells over no-restimulus background). Theresults demonstrate that a pool of Tregitopes downregulates in vitroeffector responses to co-administered peptide epitopes derived fromBotulinum neurotoxin, a protein used to treat dystonia. PBMCs from asubject with evidence of inhibitors (anti-BoNT antibodies) were culturedfor 8 days with or without a pool of Tregitope peptides (Tregitope-167,Tregitope-134, Tregitope-289). Cells were harvested and washed with PBS.2.5×10⁵ cells were re-stimulated in an IFN-γ ELISpot plate withindividual BoNT peptides, a pool of BoNT peptides, PHA positive control(not shown) or no-stimulus control. Peptides for which there was nosignificant baseline response are not shown. Response to positivecontrol PHA was robust following both culture conditions.

FIG. 22 illustrates one embodiment of a chimeric protein where apseudo-protein of interest is a string of immunogenic T cell epitopesderived from the Epstein Barr Virus (EBV) fused to a modified Fc proteinin which the Tregitope has been modified to no longer bind MHC class IImolecules and can not stimulate natural regulatory T cells.EBV-Tregitope modified Fc SEQUENCE (Kb SIGNAL SEQUENCE) is designated asunderlined text. The Tregitope is designated as bold text. The Tregitopemodified amino acids are designated as shaded text. The human Fc regionis designated as italicized text.

DETAILED DESCRIPTION General

The adaptive immune cascade begins when soluble protein antigens aretaken up by Antigen Presenting Cells (APCs) and processed through theClass II antigen presentation pathway. Protein antigens in the Class IIpresentation pathway are degraded by various proteases found in theEndoplasmic Reticulum. Some of the resulting protein fragments are boundto Class II MHC molecules. Peptide-loaded MHC molecules are traffickedto the cell surface where they are interrogated by CD4+ T cells. Peptidefragments that are capable of binding to an MHC molecule and mediatingthe cell to cell interaction between APC and circulating T cells arereferred to as T cell epitopes. Recognition of these peptide-MHCcomplexes by CD4+ T cells can lead to either an immune activating orimmune suppressive response based on the phenotype of the responding Tcells and the local cytokine/chemokine milieu. In general, engagementbetween the MHC/peptide complex and the T cell receptor (TCR) of Teffector cells leads to activation and the secretion of pro-inflammatorycytokines such as IL-4, and IFN-γ. On the other hand the activation ofnatural T regulatory cells (TReg) leads to the expression of the immunesuppressive cytokines IL-10 and TGF-β, among others (Shevach, E., Nat.Rev. Immunol., 2:389-400, 2002). These cytokines act directly on nearbyeffector T cells leading in some cases to anergy or apoptosis. In othercases regulatory cytokines and chemokines convert effector T cells to Tregulatory phenotypes; this process is referred here as “induced” or“adaptive” tolerance. T cell epitopes that are capable of binding to MHCmolecules and engaging and activating circulating Treg are referred toas Tregitopes.

Initial self/non-self discrimination occurs in the thymus duringneonatal development where medullary epithelial cells express specificself protein epitopes to immature T cells. T cells recognizing selfantigens with high affinity are deleted, but autoreactive T cells withmoderate affinity sometimes avoid deletion and can be converted to socalled natural regulatory T cells (TReg) cells. These natural TReg cellsare exported to the periphery and provide for constant suppression ofautoimmunity. Natural regulatory T cells are a critical component ofimmune regulation and self tolerance.

Self tolerance is regulated by a complex interplay between T cells, Bcells, cytokines and surface receptors. T regulatory immune responsescounterbalance T effector immune response to protein antigens (whetherself or foreign). A tilt of the balance toward the autoreactive side,either by increasing the number or function of autoreactive T effectorcells or by diminishing the number or function of T regulatory cells, ismanifested as autoimmunity.

A second form of tolerance occurs in the periphery where mature T cellsare converted to an ‘adaptive’ TReg phenotype upon activation via theirT cell receptor in the presence of IL-10 and TGF-β, usually supplied bybystander T regulatory cells. The possible roles for these ‘adaptive’TReg cells include dampening immune response following the successfulclearance of an invading pathogen as a means of controlling excessiveinflammation as might be caused by an allergic reaction or low levelchronic infection, or possibly to facilitate co-existence withbeneficial symbiotic bacteria and viruses. ‘Adaptive’ TReg may also playa role in managing the life cycle of human antibodies that haveundergone somatic hypermutation.

It is thought the constant region of immunoglobulin contains severalimportant Tregitopes whose primary function is to suppress immuneresponse to hypermutated CDRs. Due to the high volumes of circulatingIgG it is likely that there are also high volumes of T regulatory cellscorresponding to the Tregitopes contained in IgG. As a partial proof ofthis assertion consider that chimeric proteins comprising an Fc portionof an immunoglobulin bestow several desirable properties on a chimericprotein including increased stability, increased serum half life,binding to Fc receptors, and reduced immunogenicity (Lei, T. et al.,Cell. Immunol., 235:12-20, 2005, Baxevanis, C. et al., Eur. J. Immunol.,16:1013-1016, 1986).

TReg cells are also instrumental in B cell tolerance. B cells express asingle low affinity Fc receptor, FcγRIIB on their cell surface (Ravetch,J. et al., Science, 234:718-725, 1986). This receptor contains theimmunoreceptor tyrosine-based inhibition motif sequence (ITIM) in itscytoplasmic domain. Co-ligation of FCyRIIB and the BCR by immunecomplexes act to trigger the tyrosine phosphorylation of the ITIMleading to the recruitment of the inositol phosphatase, SHIP, whichinhibits BCR-triggered proliferation by interfering with the activationof MAP kinases and blocks phagocytosis by the dissociation of Burton'styrosine kinase (Btk) from the cell membrane, which inhibits calciuminflux into the cell. FcγRIIB can also induce apoptosis independent ofthe ITIM. Upon homo-aggregation of FcRIIB by ICs, the association of Btkwith the cell membrane is enhanced triggering an apoptotic response(Pearse, R. et al., Immunity, 10:753-760, 1999). Expression of FcγRIIBis highly variable and cytokine dependent. IL-4 and IL-10, which areexpressed by activated Th2 and TReg cells, have been shown to actsynergistically to enhance FcγRIIB expression (Joshi, T. et al., Mol.Immunol., 43:839-850, 2006) thus aiding in the suppression of a humoralresponse.

It is possible to exploit Tregitope specific TReg cells to suppressunwanted immune responses and to induce adaptive TReg to co-deliveredproteins. This discovery has implications for the design of therapeuticregimens and antigen-specific therapies for transplantation, proteintherapeutics, allergy, chronic infection, autoimmunity and vaccinedesign. Administration of a drug, a protein, or an allergen inconjunction with Tregitope can suppress effector immune response.Tregitope can be used to deliberately manipulate the immune systemtoward tolerance.

The peptides of the current invention are useful in the selectiveengagement and activation of regulatory T cells. It is demonstratedherein that certain pre-existing populations of regulatory T cells canbe engaged, activated and applied to the suppression of unwanted immuneresponses in both systemic and limited, disease specific, contexts.

Despite extensive efforts, with few exceptions, the antigen specificityof natural Tregs, and more importantly natural Tregs circulating inclinically significant volumes, is unknown. Presented herein is ademonstration that certain human proteins circulating in the bloodsteam, such as immunoglobulins or the serum protein Albumin, contain Tcell epitopes that relate to naturally occurring populations ofregulatory T cells. In the course of normal immune surveillance theseproteins are taken up by professional APC such as dendritic cells ormacrophages and degraded. During the degradation process some of theepitopes contained in these proteins are bound to MHC molecules,transported to the cell surface presented to regulatory T cells. Thosecells, once activated by the APC, release cytokines and chemokines helpto suppress autoimmune responses that would otherwise hinder thefunction of the extra cellular proteins.

By using the peptides of the invention to selectively activate thesepre-existing regulatory T cells, it is herein shown that the peptides ofthe invention can be used to suppress a variety of unwanted immuneresponses. In its simplest form systemic application of the peptides ofthe invention can be used as a generalized immune suppressant useful forcontrolling severe autoimmune reactions such as, for example, MSflare-ups, allergic reactions, transplant reactions, or uncontrolledresponse to infection. In a more controlled application, topicallyapplied to joints affected by rheumatoid arthritis (RA) for example, thepeptide of the invention can be used to suppress localized autoimmuneresponses. In a targeted application, such as might be achieved throughthe fusion or bonding of the peptides to certain other T cell epitopes,the peptides can suppress highly specific immune reactions while leavingthe balance of the immune system intact. For example, through thedelivery of a regulatory peptide fused to an autoimmune antigen such asinsulin, or an allergen such as Brazil nut antigen, the immune systemcan be trained to “tolerate” the co-delivered antigen by converting thephenotype of responding effector T cells to that of adaptive regulatoryT cells.

As stated above the peptides of the current invention are derived fromcirculating extracellular proteins. To be useful these peptides must betrue T cell epitopes capable of binding to both MHC molecules and TCRs),and be related to a pre-existing population of regulatory T cells thatis sufficiently large to have a therapeutic affect. T cell epitopeclusters, epitopes capable of binding to multiple MHC alleles andmultiple TCRs, are key to satisfying this latter qualification.

Definitions

To further facilitate an understanding of the present invention, anumber of terms and phrases are defined below.

As used herein, the term “biological sample” as refers to any sample oftissue, cells or secretions from an organism.

As used herein, the term “transplantation” refers to the process oftaking a cell, tissue, or organ, called a “transplant” or “graft” fromone subject and placing it or them into a (usually) different subject.The subject who provides the transplant is called the “donor”, and thesubject who received the transplant is called the “recipient”. An organor graft transplanted between two genetically different subjects of thesame species is called an “allograft”. A graft transplanted betweensubjects of different species is called a “xenograft”.

As used herein, the term “medical condition” includes, but is notlimited to, any condition or disease manifested as one or more physicaland/or psychological symptoms for which treatment and/or prevention isdesirable, and includes previously and newly identified diseases andother disorders.

As used herein, the term “immune response” refers to the concertedaction of lymphocytes, antigen presenting cells, phagocytic cells,granulocytes, and soluble macromolecules produced by the above cells orthe liver (including antibodies, cytokines, and complement) that resultsin selective damage to, destruction of, or elimination from the humanbody of cancerous cells, metastatic tumor cells, malignant melanoma,invading pathogens, cells or tissues infected with pathogens, or, incases of autoimmunity or pathological inflammation, normal human cellsor tissues.

As used herein, the term “effective amount” of a composition, is aquantity sufficient to achieve a desired therapeutic and/or prophylacticeffect, e.g., an amount that results in the prevention of, or a decreasein, the symptoms associated with a disease that is being treated. Theamount of a composition of the invention administered to the subjectwill depend on the type and severity of the disease and on thecharacteristics of the individual, such as general health, age, sex,body weight and tolerance to drugs. It will also depend on the degree,severity and type of disease. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors. Thecompositions of the present invention can also be administered incombination with each other or with one or more additional therapeuticcompounds.

As used herein, the term “T cell epitope” means a protein determinant, 7to 30 amino acids in length, and capable of specific binding to HLAmolecules and interacting with specific TCRs. Generally, T cell epitopesare linear and do not express specific three dimensionalcharacteristics. T cell epitopes are not affected by the presence ofdenaturing solvents.

As used herein, the term “B cell epitope” means a protein determinantcapable of specific binding to an antibody. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

The term “subject” as used herein refers to any living organism in whichan immune response is elicited. The term subject includes, but is notlimited to, humans, nonhuman primates such as chimpanzees and other apesand monkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

As used herein, the term MHC complex refers to a protein complex capableof binding with a specific repertoire of polypeptides known as HLAligands and transporting said ligands to the cell surface.

As used herein, the term “MHC Ligand” means a polypeptide capable ofbinding to one or more specific MHC alleles. The term “HLA ligand” isinterchangeable with the term MHC Ligand. Cells expressing MHC/Ligandcomplexes on their surface are referred to as “Antigen Presenting Cells”(APCs).

As used herein, the term T Cell Receptor or TCR refers to a proteincomplex expressed by T cells that is capable of engaging a specificrepertoire of MHC/Ligand complexes as presented on the surface of APCs.

As used herein, the term “T cell epitope” means an MHC ligand capable ofinteracting with specific T cell receptors (TCRs). T cell epitopes canbe predicted by in silico methods (De Groot, A. et al., AIDS Res. Hum.Retroviruses, 13:539-541, 1997; Schafer, J. et al., Vaccine,16:1880-1884, 1998; De Groot, A. et al., Vaccine, 19:4385-95, 2001; DeGroot, A. et al., Vaccine, 21:4486-504, 2003).

As used herein, the term “MHC Binding Motif” refers to a pattern ofamino acids in a protein sequence that predicts binding to a particularMHC allele.

As used herein, the term “T-cell epitope cluster” refers to polypeptidethat contains between about 4 to about 40 MHC binding motifs. Inparticular embodiments, the T-cell epitope cluster contains betweenabout 5 to about 35 MHC binding motifs, between about 8 and about 30 MHCbinding motifs; and between about 10 and 20 MHC binding motifs.

As used herein, the term “EpiBar” refers to a single 9-mer frame that ispredicted to be reactive to at least four different HLA alleles.Sequences of known immunogens that contain EpiBars include InfluenzaHemagglutinin 307-319, Tetanus Toxin 825-850, and GAD65 557-567. Anexample of an immunogenic peptide that contains an EpiBar is shown inFIG. 17. FIG. 17 depicts an example of an EpiBar and the EpiMatrixanalysis of a pormiscous influenza epitope. Consider the influenza HApeptide, an epitope known to be promiscuously immunogenic. It scoresextremely high for all eight alleles in EpiMatrix. Its cluster score is18. Cluster scores higher than 10 are considered to be significant. Theband-like EpiBar pattern is characteristic of promiscuous epitopes.Results are shown in FIG. 17 for PRYVKQNTL (SEQ ID NO:59), RYVKQNTLK(SEQ ID NO:60), YVKQNTLKL (SEQ ID NO:61), VKQNTLKLA (SEQ ID NO:62) andKQNTLKLAT (SEQ ID NO:63). Z score indicates the potential of a 9-merframe to bind to a given HLA allele. All scores in the top 5% areconsidered “hits”, while non hits (*) beow 10% are masked in FIG. 17 forsimplicity.

As used herein, the term “Immune Synapse” means the protein complexformed by the simultaneous engagement of a given T cell Epitope to botha cell surface MHC complex and TCR.

As used herein, the term “regulatory T cell” means a subset of naturallyoccurring T cells characterized by the presence of certain cell surfacemarkers including but not limited to CD4, CD25, and FoxP3. Uponactivation regulatory T cells secrete immune suppressive cytokines andchemokines including but not limited to IL-10, TGF-β and TNF-α.

The term “polypeptide” refers to a polymer of amino acids, and not to aspecific length; thus, peptides, oligopeptides and proteins are includedwithin the definition of a polypeptide. As used herein, a polypeptide issaid to be “isolated” or “purified” when it is substantially free ofcellular material when it is isolated from recombinant andnon-recombinant cells, or free of chemical precursors or other chemicalswhen it is chemically synthesized. A polypeptide, however, can be joinedto another polypeptide with which it is not normally associated in acell and still be “isolated” or “purified.” When a polypeptide isrecombinantly produced, it can also be substantially free of culturemedium, for example, culture medium represents less than about 20%, lessthan about 10%, or less than about 5% of the volume of the polypeptidepreparation.

A variant polypeptide can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these.

The invention also includes polypeptide fragments of the polypeptides ofthe invention. The invention also encompasses fragments of the variantsof the polypeptides described herein. The invention also provideschimeric or fusion polypeptides. These comprise a polypeptide of theinvention operatively linked to a heterologous protein or polypeptidehaving an amino acid sequence not substantially homologous to thepolypeptide. “Operatively linked” indicates that the polypeptide and theheterologous protein are fused in-frame.

The isolated polypeptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Inone embodiment, the polypeptide is produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding thepolypeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the polypeptide expressed in the hostcell. The polypeptide can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques.

For the purposes of the present invention, polypeptides can include, forexample, modified forms of naturally occurring amino acids such asD-stereoisomers, non-naturally occurring amino acids; amino acidanalogs; and mimetics.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and the Claims. In the Specification and the appendedClaims, the singular forms include plural referents unless the contextclearly dictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. All references cited herein are incorporated herein byreference in their entirety and for all purposes to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Compositions

In one aspect, the invention provides a novel class of T cell epitopescompositions, termed ‘Tregitopes’, which comprise a peptide orpolypeptide chain with one or more defined characteristics listed below.That is, the Tregitopes of the invention include, but are not limitedto, possessing one or more of the following characteristics:

(1) Tregitopes of the invention are derived from common human proteins.(2) Tregitopes of the invention are highly conserved among knownvariants of their source proteins (e.g., present in more than 50% ofknown variants).(3) Tregitopes of the invention comprise at least one putative T cellepitope as identified by EpiMatrix analysis. EpiMatrix is a proprietarycomputer algorithm developed by EpiVax, which is used to screen proteinsequences for the presence of putative T cell epitopes. Input sequencesare parsed into overlapping 9-mer frames where each frame overlaps thelast by 8 amino acids. Each of the resulting frames is then scored forpredicted binding affinity with respect to a panel of eight common ClassII HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801,DRB1*1101, DRB1*1301, and DRB1*1501). Raw scores are normalized againstthe scores of a large sample of randomly generated peptides. Theresulting “Z” score is reported. Any 9-mer peptide with anallele-specific EpiMatrix Z-score in excess of 1.64, theoretically thetop 5% of any given sample, is considered a putative T cell epitope.

In a preferred embodiment the Tregitopes of the invention containseveral putative T cell epitopes forming a pattern known as a T cellepitope cluster. Putative T-cell epitopes are not randomly distributedthroughout protein sequences but instead tend to “cluster” in specificregions. In addition, peptides containing clusters of putative T cellepitopes are more likely to test positive in validating in vitro and invivo assays. The results of the initial EpiMatrix analysis are furtherscreened for the presence of putative T cell epitope “clusters” using asecond proprietary algorithm known as Clustimer. The Clustimer algorithmidentifies sub-regions contained within any given amino acid sequencethat contains a statistically unusually high number of putative T cellepitopes. Typical T-cell epitope “clusters” range from about 9 toroughly 30 amino acids in length and, considering their affinity tomultiple alleles and across multiple 9-mer frames, can contain anywherefrom about 4 to about 40 putative T cell epitopes. For each epitopecluster identified an aggregate EpiMatrix score is calculated by summingthe scores of the putative T cell epitopes and subtracting a correctingfactor based on the length of the candidate epitope cluster and theexpected score of a randomly generated cluster of the same length.EpiMatrix cluster scores in excess of +10 are considered significant.

Many of the most reactive T cell epitope clusters contain a featurereferred to as an “EpiBar”. An EpiBar is a single 9-mer frame that ispredicted to be reactive to at least four different HLA alleles.Sequences that contain EpiBars include Influenza Hemagglutinin 307-319(Cluster score of 18), Tetanus Toxin 825-850 (Cluster score of 16), andGAD65 557-567 (Cluster score of 19). In another embodiment, the peptidesof the invention can comprise oen or more EpiBars.

(4) Tregitopes of the invention bind to at least one and preferably twoor more common HLA class II molecules with at least a moderate affinity(e.g., <200 μM IC₅₀ in HLA binding assays based on soluble HLAmolecules).(5) Tregitopes of the invention are capable of being presented at thecell surface by APCs in the context of at least one and, in a preferredembodiment, two or more alleles of the HLA.(6) In this context, the Tregitope-HLA complex can be recognized bypre-existing populations of regulatory T cells having TCRs that arespecific for the Tregitope-HLA complex and circulating in normal controlsubjects. The recognition of the Tregitope-HLA complex can cause thematching regulatory T cell to be activated and to secrete regulatorycytokines and chemokines.(7) Stimulating regulatory T cells with Tregitope(s) of the inventionresults in increased secretion of one or more of the following cytokinesand chemokines: IL-10, TGF-8, TNF-α and MCP1. This increased secretionof regulatory cytokines and chemokines is a hallmark of regulatory Tcells.(8) Regulatory T cells activated by the Tregitope(s) of the inventionexpress a CD4+CD25+FOXP3 phenotype.(9) Regulatory T cells activated by the Tregitope(s) directly suppressT-effector immune responses ex vivo as measured by decreasedantigen-specific Th1- or Th2-associated cytokine levels, principallyINF-γ, IL-4, and IL-5, and by decreased proliferation ofantigen-specific T effector cells as measured by CFSE dilution.(10) Regulatory T cells activated by the Tregitope(s) directly suppressT effector immune responses in vivo as measured by decreasedantigen-specific Th1- or Th2-associated cytokine levels (as measured byElisa assay), decreased antigen-specific T effector cell levels (asmeasured by EliSpot assay) and decreased antibody titers for proteinantigens.(11) Natural regulatory T cells activated by the Tregitopes of theinvention stimulate the development of adaptive T_(Reg) cells.Co-incubating peripheral T cells with the Tregitopes of the invention inthe presence of antigen results in the expansion of antigen-specificCD4+/CD25+ T cells, upregulates the expression of FOXP3+ on those cellsand suppresses the activation of antigen-specific T effector cells invitro.

The Tregitopes of the invention are useful for regulating immuneresponse to monoclonal antibodies, protein therapeutics, self antigenspromoting autoimmune response, allergens, transplanted tissues and inother applications where tolerance is the desired outcome. Selectembodiments of the Tregitopes of the invention are summarized in FIG.18A and FIG. 18B in Example 1.

In one embodiment, the Tregitope of the invention is a T cell epitopeisolated as decribed in FIG. 18A and FIG. 18B. The Tregitopes of FIG.18A and FIG. 18B (SEQ ID NOs: 4 through 58) can bind MHC class IImolecules, engage TCR in context of MHC class II molecules and activatenatural regulatory T cells.

The polypeptides of the invention can be purified to homogeneity orpartially purified. It is understood, however, that preparations inwhich the polypeptide is not purified to homogeneity are useful. Thecritical feature is that the preparation allows for the desired functionof the polypeptide, even in the presence of considerable amounts ofother components. Thus, the invention encompasses various degrees ofpurity. In one embodiment, the language “substantially free of cellularmaterial” includes preparations of the polypeptide having less thanabout 30% (by dry weight) other proteins (e.g., contaminating protein),less than about 20% other proteins, less than about 10% other proteins,or less than about 5% other proteins.

When a polypeptide is recombinantly produced, it can also besubstantially free of culture medium, for example, culture mediumrepresents less than about 20%, less than about 10%, or less than about5% of the volume of the polypeptide preparation. The language“substantially free of chemical precursors or other chemicals” includespreparations of the polypeptide in which it is separated from chemicalprecursors or other chemicals that are involved in its synthesis. Thelanguage “substantially free of chemical precursors or other chemicals”can include, for example, preparations of the polypeptide having lessthan about 30% (by dry weight) chemical precursors or other chemicals,less than about 20% chemical precursors or other chemicals, less thanabout 10% chemical precursors or other chemicals, or less than about 5%chemical precursors or other chemicals.

As used herein, two polypeptides (or a region of the polypeptides) aresubstantially homologous or identical when the amino acid sequences areat least about 45-55%, typically at least about 70-75%, more typicallyat least about 80-85%, and more typically greater than about 90% or morehomologous or identical. To determine the percent homology or identityof two amino acid sequences, or of two nucleic acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the sequence of one polypeptide or nucleic acid moleculefor optimal alignment with the other polypeptide or nucleic acidmolecule). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in one sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the other sequence, then themolecules are homologous at that position. As used herein, amino acid ornucleic acid “homology” is equivalent to amino acid or nucleic acid“identity”. The percent homology between the two sequences is a functionof the number of identical positions shared by the sequences (e.g.,percent homology equals the number of identical positions/total numberof positions×100).

The invention also encompasses polypeptides having a lower degree ofidentity but having sufficient similarity so as to perform one or moreof the same functions performed by a polypeptide encoded by a nucleicacid molecule of the invention. Similarity is determined by conservedamino acid substitution. Such substitutions are those that substitute agiven amino acid in a polypeptide by another amino acid of likecharacteristics. Conservative substitutions are likely to bephenotypically silent. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found, for example, in Bowie, J. et al., Science,247:1306-1310, 1990.

A variant polypeptide can differ in amino acid sequence by one or moresubstitutions, deletions, insertions, inversions, fusions, andtruncations or a combination of any of these. Variant polypeptides canbe fully functional or can lack function in one or more activities.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions can positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region. Severalexamples of variant polypeptides are included in FIG. 18A and FIG. 18B.

The invention also includes polypeptide fragments of the polypeptides ofthe invention. The invention also encompasses fragments of the variantsof the polypeptides described herein. As used herein, a fragmentcomprises at least about five contiguous amino acids. Useful fragmentsinclude those that retain one or more of the biological activities ofthe polypeptide as well as fragments that can be used as an immunogen togenerate polypeptide-specific antibodies. Biologically active fragmentsare, for example, about 6, 9, 12, 15, 16, 20 or 30 or more amino acidsin length. Fragments can be discrete (not fused to other amino acids orpolypeptides) or can be within a larger polypeptide. Several fragmentscan be comprised within a single larger polypeptide. In one embodiment afragment designed for expression in a host can have heterologous pre-and pro-polypeptide regions fused to the amino terminus of thepolypeptide fragment and an additional region fused to the carboxylterminus of the fragment.

The invention also provides chimeric or fusion polypeptides. Thesecomprise a polypeptide of the invention operatively linked to aheterologous protein or polypeptide having an amino acid sequence notsubstantially homologous to the polypeptide. “Operatively linked”indicates that the polypeptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the polypeptide. In one embodiment the fusion polypeptidedoes not affect function of the polypeptide per se. For example, thefusion polypeptide can be a GST-fusion polypeptide in which thepolypeptide sequences are fused to the C-terminus of the GST sequences.Other types of fusion polypeptides include, but are not limited to,enzymatic fusion polypeptides, for example beta-galactosidase fusions,yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions. Suchfusion polypeptides, particularly poly-His fusions or affinity tagfusions, can facilitate the purification of recombinant polypeptide. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a polypeptide can be increased by using a heterologoussignal sequence. Therefore, in another embodiment, the fusionpolypeptide contains a heterologous signal sequence at its N-terminus.

A chimeric or fusion polypeptide can be produced by standard recombinantDNA techniques. For example, DNA fragments coding for the differentpolypeptide sequences are ligated together in-frame in accordance withconventional techniques. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of nucleic acid fragmentscan be carried out using anchor primers which give rise to complementaryoverhangs between two consecutive nucleic acid fragments which cansubsequently be annealed and re-amplified to generate a chimeric nucleicacid sequence (Ausubel et al., Current Protocols in Molecular Biology,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST protein). A nucleic acidmolecule encoding a polypeptide of the invention can be cloned into suchan expression vector such that the fusion moiety is linked in-frame tothe polypeptide.

The isolated polypeptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods. Inone embodiment, the polypeptide is produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding thepolypeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the polypeptide expressed in the hostcell. The polypeptide can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques.

The invention also provides for nucleic acids that encode in whole or inpart the polypeptides of the invention. The nucleic acid molecules ofthe invention can be inserted into vectors and used, for example, asexpression vectors or gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, e.g., intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(Chen, et al., Proc. Natl. Acad. Sci. USA, 91:3054-3057, 1994). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

Tregitopes of the invention can include allelic or sequence variants(“mutants”) or analogs thereof, or can include chemical modifications(e.g., pegylation, gycosylation). In one instance, a mutant can providefor enhanced binding to MHC molecules. In another instance, a mutant canlead to enhanced binding to TCRs. In an other instance, a mutant canlead to a decrease in binding to MHC molecules and/or TCRs. Alsocontemplated is a mutant that binds but does not allow signaling via theTCR.

The invention provides for Tregitope compositions that are chimericprotein compositions. In one embodiment, the Tregitope compositioncomprises a first and a second polypeptide chain linked together,wherein the first chain comprises sequence numbers 4 through 58 or anycombination thereof, and said second chain comprises a biologicallyactive molecule. In one embodiment, the biologically active molecule isselected from the group consisting of: an immunogenic molecule; a T cellepitope; viral protein; bacterial protein. In one embodiment, theTregitope composition of the invention comprises a first and a secondpolypeptide chain linked together, wherein said first chain comprises aFc region wherein the amino acids in region 289-309 has been altered soas not to bind to MHC class II molecules, and said second chaincomprises a immunogenic molecule.

In one aspect, the invention provides methods to produce a regulatory Tcell line recognizing at least a portion of SEQ ID NOS:4-58. In oneembodiment, one or more peptides selected from the group consisting ofSEQ ID NOS:4-58 are combined in admixture with an appropriate excipient.Such compositions are useful in methods of preventing or treatinginflammation in a subject in need thereof, wherein local delivery of theadmixture with an appropriate excipient results in decreasedinflammation in the subject.

In one embodiment, one or more peptides selected from the groupconsisting of SEQ ID NOS:4-58 are combined in admixture with a antigenor allergen. Such compositions are useful in methods of inducingtolerance to the antigen or allergen in a subject in need thereof,wherein local delivery of the admixture with a antigen or allergenresults in increased tolerance to the antigen or allergen in thesubject, and delivered with an appropriate excipient resulting ininduced tolerance to the antigen or allergen.

In one embodiment, the invention provides a nucleic acid encodingcomprising one or more of the Tregitope polypeptides selected from thegroup consisting of SEQ ID NOS:4-58. In one embodiment, the inventionprovides a vector comprising a nucleic acid encoding comprising one ormore of the Tregitope polypeptides selected from the group consisting ofSEQ ID NOS:4-58. In one embodiment, the invention provides a cellcomprising a vector of the invention. The cell can be a mammalian cell,bacterial cell, insect cell, or yeast cell.

Cloning of Tregitope Specific T Cells

Cloning of Tregitope specific T cells can be conducted by techniquesknown to one of skill in the art. For example, isolated PBMCs arestimulated with Tregitopes at 10 μg/ml RPMI media containing 20% HSA.IL-2 is added (10 U/ml final concentration) every other day starting onday 5. T cells are stained with tetramer pools on day 11 or 12. For eachpool, 2-3×10⁵ cells are incubated with 0.5 mg of PE-labeled tetramer in50 ml of culture medium (10 mg/ml) at 37° C. for 1 to 2 h, and thenstained with anti-CD4-FITC (BD PharMingen, San Diego, Calif.) for 15 minat room temperature. Cells are washed and analyzed with a BectonDickinson FACSCalibur flow cytometer (Becton Dickinson, San Jose,Calif.). Tetramers loaded with the corresponding single peptides aregenerated for those pools that give positive staining, and analysis isdone on day 14 or 15. Cells that are positive for a particular tetramerare single-cell sorted into 96-well U-bottom plates by using a BectonDickinson FACSVantage (San Jose, Calif.) on the same or following day.Sorted cells are expanded with 1.5-3×10⁵ unmatched, irradiated (5000rad) PBMC per well as feeders with 2.5 mg/ml PHA and 10 U/ml IL-2 added24 h later. Specificity of cloned T cells is confirmed by staining withtetramers (loaded with cognate peptide or control peptide, HA307-319)and T cell proliferation assays with 10 mg/ml of specific peptide(Novak, E. et al., J. Immunol., 166:6665-6670, 2001).

Methods of Use of Tregitope Compositions

In one aspect, the invention provides methods of using Tregitopes forthe purpose of designing small molecules. In one method of theinvention, Tregitope-specific T cells are stimulated three times withpools of small molecule mixtures at a concentration of 1 μg/ml andautologous dendritic cells (DC) at 2-week intervals, followed bystimulation with heterologous DC and antigens. T cells (1.25×10⁵) and DC(0.25×10⁵) are added per well in round-bottom, 96-well plates. T cellmedium is made by supplementing 500 ml of RPMI medium 1640 with 50 ml ofFCS (HyClone), penicillin, and streptomycin (GIBCO); 20 mM Hepes(GIBCO); and 4 ml 1 N NaOH solution. The IL-2 concentration is initially0.1 nM and gradually is increased to 1 nM during subsequent rounds ofstimulation. T cell clones are derived by limiting dilution by using0.6×10⁵ Epstein-Barr virus-transformed B cells (100 Gray) and 1.3×10⁵heterologous peripheral blood mononuclear cells (33 Gray) as feedercells and 1 μg/ml phytohemagglutinin (Difco) in medium containing 2 nMIL-2. Small molecules pools that stimulate the Tregitope specific Tcells are then tested as individual molecules.

In one aspect, the invention provides methods of using Tregitopes forthe purpose of cloning T cell receptors. Total RNA is extracted with anRNeasy Mini Kit (Qiagene) from the Tregitope specific T cell linesgenerated as described above. One microgram of total RNA is used toclone the TCR cDNAs by a rapid amplification of cDNA end (RACE) method(GeneRacer Kit, Invitrogen). Before synthesizing the single-strand cDNA,the RNA is dephosphorylated, decapped, and ligated with an RNAoligonucleotide according to the instruction manual of 5′ RACE GeneRacerKit. SuperScript II RT and GeneRacer Oligo-dT are used for reversetranscription of the RNA Oligo-ligated mRNA to single-strand cDNAs. 5′RACE is performed by using GeneRacer 5′ (GeneRacer Kit) as 5′ primer andgene-specific primer TCRCAR (5′-GTT AAC TAG TTC AGC TGG ACC ACA GCC GCAGC-3′; SEQ ID NO:64) or TCRCB1R (5′-CGG GTT AAC TAG TTC AGA AAT CCT TTCTCT TGA CCA TGG C-3′; SEQ ID NO:65), or TCRCBR2 (5′-CTA GCC TCT GGA ATCCTT TCT CTT G-3′; SEQ ID NO:66) as 3′ primers for TCR α, β1, or β2chains, respectively. The polymerase chain reaction (PCR) products arecloned into pCR2.1 TOPO vector (Invitrogen) and then transformed intoOne Shot TOP10 Competent Escherichia coli (Invitrogen). Plasmid DNAs areprepared from 96 individual clones from each construct for TCRα, β1, andβ2 chains. Full-length insert of all the plasmids is sequenced todetermine the va/v13 usage (Zhao, Y. et al., J. Immunother., 29:398-406,2006).

Pharmaceutical Formulations

The invention provides methods of treating a subject with a medicalcondition comprising administering a therapeutically effective amount ofa Tregitope in a pharmaceutically acceptable carrier or excipient. TheTregitopes of the present invention can be incorporated intopharmaceutical compositions suitable for administration. Thepharmaceutical compositions generally comprise at least one Tregitopeand a pharmaceutically-acceptable carrier in a form suitable foradministration to a subject. Pharmaceutically-acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations ofpharmaceutical compositions for administering the Tregitope compositions(see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa. 18th ed., 1990). The pharmaceutical compositions aregenerally formulated as sterile, substantially isotonic and in fullcompliance with all Good Manufacturing Practice (GMP) regulations of theU.S. Food and Drug Administration.

The terms “pharmaceutically-acceptable,” “physiologically-tolerable,”and grammatical variations thereof, as they refer to compositions,carriers, diluents and reagents, are used interchangeably and representthat the materials are capable of administration to or upon a subjectwithout the production of undesirable physiological effects to a degreethat would prohibit administration of the composition.“Pharmaceutically-acceptable excipient” means, for example, an excipientthat is useful in preparing a pharmaceutical composition that isgenerally safe, non-toxic, and desirable, and includes excipients thatare acceptable for veterinary use as well as for human pharmaceuticaluse. Such excipients can be solid, liquid, semisolid, or, in the case ofan aerosol composition, gaseous. A person of ordinary skill in the artwould be able to determine the appropriate timing, sequence and dosagesof administration for particular drugs and compositions of the presentinvention.

Preferred examples of such carriers or diluents include, but are notlimited to, water, saline, Ringer's solutions, dextrose solution, and 5%human serum albumin. Liposomes and non-aqueous vehicles such as fixedoils can also be used. The use of such media and compounds forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or compound is incompatible with theTregitope, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. The Tregitopecompositions of the present invention can be administered by parenteral,topical, intravenous, oral, subcutaneous, intraarterial, intradermal,transdermal, rectal, intracranial, intraperitoneal, intranasal;vaginally; intramuscular route or as inhalants. In some embodiments ofthe invention, agents are injected directly into a particular tissuewhere deposits have accumulated, e.g., intracranial injection.Intramuscular injection or intravenous infusion are preferred foradministration of the Tregitope. In some methods, particular Tregitopesof the invention are injected directly into the cranium. In somemethods, the Tregitopes of the invention are administered as a sustainedrelease composition or device, such as a Medipad™ device.

The Tregitope of the invention can optionally be administered incombination with other agents that are at least partly effective intreating various medical conditions as described herein. In the case ofadministration into the central nervous system of a subject, theTregitope of the invention can also be administered in conjunction withother agents that increase passage of the agents of the invention acrossthe blood-brain barrier.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial compounds such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating compounds such as ethylenediaminetetraacetic acid (EDTA);buffers such as acetates, citrates or phosphates, and compounds for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. Examples of excipients can include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol,DMSO, glycol, propylene, dried skim milk, and the like. The compositioncan also contain pH buffering reagents, and wetting or emulsifyingagents.

The parenteral preparation can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition is sterile and should be fluid to the extent thateasy syringeability exists. It is stable under the conditions ofmanufacture and storage and is preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, e.g., water, ethanol, polyol(e.g., glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, e.g., by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prevention of the action of microorganismscan be achieved by various antibacterial and antifungal compounds, e.g.,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, it will be preferable to include isotoniccompounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition acompound that delays absorption, e.g., aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating theTregitope in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the binding agent into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The agents of this invention can be administered inthe form of a depot injection or implant preparation that can beformulated in such a manner as to permit a sustained or pulsatilerelease of the active ingredient.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the bindingagent can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding compounds, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating compound such as alginicacid, Primogel or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningcompound such as sucrose or saccharin; or a flavoring compound such aspeppermint, methyl salicylate or orange flavoring.

For administration by inhalation, the Tregitope(s) are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, e.g., fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the Tregitope is formulated into ointments, salves, gels, or creams andapplied either topically or through transdermal patch technology asgenerally known in the art.

The Tregitope can also be prepared as pharmaceutical compositions in theform of suppositories (e.g., with conventional suppository bases such ascocoa butter and other glycerides) or retention enemas for rectaldelivery.

In one embodiment, the Tregitope is prepared with carriers that protectthe Tregitope against rapid elimination from the body, such as acontrolled-release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as, for example, ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used aspharmaceutically-acceptable carriers. These can be prepared according tomethods known to those skilled in the art (U.S. Pat. No. 4,522,811). TheTregitopes or chimeric proteins can be implanted within or linked to abiopolymer solid support that allows for the slow release of theTregitopes or chimeric proteins to the desired site.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of bindingagent calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the binding agent and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such Tregitope for the treatment of asubject.

Methods of Preventing or Treating a Medical Condition

The present invention is directed to, for example methods of treatingone or more medical conditions comprising administering a Tregitope orchimeric protein of the invention, thereby treating the medicalcondition. The medical condition can be, for example, primaryimmunodeficiencies; immune-mediated thrombocytopenia, Kawasaki disease,hematopoietic stem cell transplantation in patients older than 20 years,chronic B-cell lymphocytic leukemia and pediatric HIV type 1 infections.Specific examples include: (Hematology) aplastic anemia, pure red cellaplasia, Diamond-Blackfan anemia, autoimmune hemolytic anemia, hemolyticdisease of the newborn, acquired factor VIII inhibitors, acquired vonWillebrand disease, immune-mediated neutropenia, refractoriness toplatelet transfusion, neonatal alloimmune/autoimmune thrombocytopenia,posttransfusion purpura, thrombotic thrombocytopenia purpura/hemolyticuremic syndrome; (Infectious diseases) conditions in which acquiring aninfectious disease could be deleterious include low birth weight (e.g.,<1500 g), solid organ transplantation, surgery, trauma, burns, and HIVinfection; (Neurology) epilepsy and pediatric intractable Guillain-Barrésyndrome, chronic inflammatory demyelinating polyneuropathy, myastheniagravis, Lambert-Eaton myasthenic syndrome, multifocal motor neuropathy,multiple sclerosis; (Obstetrics) recurrent pregnancy loss; (Pulmonology)asthma, chronic chest symptoms, rheumatology, rheumatoid arthritis(adult and juvenile), systemic lupus erythematosus, systemicvasculitides, dermatomyositis, polymyositis, inclusion-body myositis,wegener granulomatosis; (Miscellaneous) adrenoleukodystrophy,amyotrophic lateral sclerosis, Behcet syndrome, acute cardiomyopathy,chronic fatigue syndrome, congential heart block, cystic fibrosis,autoimmune blistering dermatosis, diabetes mellitus, acute idiopathicdysautonomia, acute disseminated encephalomyelitis, endotoxemia,hemolytic transfusion reaction, hemophagocytic syndrome, acutelymphoblastic leukemia, lower motor neuron syndrome, multiple myeloma,human T-cell lymphotrophic virus-1-associated myelopathy, nephriticsyndrome, membranous nephropathy, nephrotic syndrome, euthyroidophthalmopathy, opsoclonus-myoclonus, recurrent otitis media,paraneoplastic cerebellar degeneration, paraproteinemic neuropathy,parvovirus infection (general), polyneuropathy, organomegaly,endocrinopathy, M-protein, and skin changes (POEMS) syndrome,progressive lumbosacral plexopathy, lyme radiculoneuritis, Rasmussensyndrome, Reiter syndrome, acute renal failure, thrombocytopenia(nonimmune), streptococcal toxic shock syndrome, uveitis andVogt-Koyanagi-Harada syndrome.

In particular embodiment, the present invention is directed to, forexample, methods of treating allergy, autoimmune disease,transplant-related disorders such as graft versus host disease, enzymeor protein deficiency disorders, hemostatic disorders, cancers,infertility, or infections (viral, bacterial, or parasitic). TheTregitopes or chimeric proteins of the invention can be used with inconjunction with other proteins or compounds used for treating a subjectwith a medical condition in order to reduce adverse events or enhancethe efficacy of the co-administered compound.

Application to Allergy. Allergen-specific regulatory T cells play animportant role in controlling the development of allergy and asthma.Both naturally occurring CD4/CD25 regulatory T cells and secondaryT_(Regs) (antigen-specific regulatory T cells), both expressing thetranscription factor FOXp3, have been shown to inhibit the inappropriateimmune responses involved in allergic diseases. A number of recentstudies indicate that regulatory T cells play an important role incontrolling the overdevelopment of T-helper type 2 biased immuneresponses in susceptible individuals, not only in animal models, but inhumans as well. Recent studies indicate that T regulatory cells alsosuppress T cell costimulation by the secretion of TGF-β and IL-10,suggesting an important role of T regulatory cells in the regulation ofallergic disorders. Impaired expansion of natural or adaptive regulatoryT cells leads to the development of allergy, and treatment to induceallergen-specific regulatory T cells would provide curative therapiesfor allergy and asthma.

One strategy both for the prevention and therapy of asthma is theinduction of regulatory T cells. Animals can be protected fromdeveloping asthma by immune stimulation leading to Th1 or Tr responses.

Application to Transplantation. The Tregitopes of the invention areuseful to induce tolerance during the transplantation process, bypromoting the development of cells that specifically down regulateimmune responses against donor cells. Induction of Ag-specific T_(Reg)cells for treating organ-specific autoimmunity is an importanttherapeutic development, avoiding generalized immune suppression. Inmurine models of bone marrow transplantation, T_(Regs) promote donorbone marrow engraftment and decrease the incidence and severity of graftversus host disease without abrogating the beneficial graft versus tumorimmunologic effect. These findings, in concert with observations thatT_(Regs) in mice and humans share phenotypic and functionalcharacteristics, have led to active investigations into the use of thesecells to decrease complications associated with human hematopoietic celltransplantation. An imbalance of T_(Regs) and effector T cellscontributes to the development of graft versus host disease. However,the mechanisms of immunoregulation, in particular the allorecognitionproperties of T_(Regs), their effects on and interaction with otherimmune cells, and their sites of suppressive activity, are not wellunderstood.

Accumulating evidence from both humans and experimental animal modelshas implicated the involvement of T_(Regs) in the development of graftversus host disease (GVHD). The demonstration that T_(Regs) can separateGVHD from graft versus tumor (GVT) activity suggests that theirimmunosuppressive potential could be manipulated to reduce GVHD withoutdetrimental consequence on GVT effect. Although a variety of Tlymphocytes with suppressive capabilities have been reported, the twobest-characterized subsets are the naturally arising,intrathymic-generated T_(Regs) (natural TRe_(g)s) and the peripherallygenerated, inducible T_(Regs) (inducible TRe_(g)s).

Application to Autoimmunity. Tregitopes can be used as a tolerizingagents for immunogenic compounds (protein therapeutics). This discoveryhas implications for the design of protein therapeutics. Thus,administration of a monoclonal antibody, autologous cytokine, or foreignprotein in conjunction with Tregitopes suppresses adverse T effectorimmune responses. In vivo, T_(Regs) act through dendritic cells to limitautoreactive T-cell activation, thus preventing their differentiationand acquisition of effector functions. By limiting the supply ofactivated pathogenic cells, T_(Regs) prevent or slow down theprogression of autoimmune diseases. This protective mechanism appears,however, insufficient in autoimmune individuals, likely because of ashortage of T_(Regs) cells and/or the development and accumulation ofT_(Reg)-resistant pathogenic T cells over the long disease course. Thus,restoration of self-tolerance in these patients may require purging ofpathogenic T cells along with infusion of T_(Regs) with increasedability to control ongoing tissue injury. Organ-specific autoimmuneconditions, such as thyroiditis and insulin-dependent diabetes mellitushave been attributed to a breakdown of this tolerance mechanism.

Application to Diabetes. Type 1 (juvenile) diabetes is an organ-specificautoimmune disease resulting from destruction of insulin-producingpancreatic beta-cells. In non-diabetics, islet cell antigen-specific Tcells are either deleted in thymic development or are converted to Tregulatory cells that actively suppress effector responses to islet cellantigens. In juvenile diabetics and in the NOD mouse model of juvenilediabetes, these tolerance mechanisms are missing. In their absence,islet cell antigens are presented by human leukocyte antigen (HLA) classI and II molecules and are recognized by CD8(+) and CD4(+) auto-reactiveT cells. Destruction of islet cells by these auto-reactive cellseventually leads to glucose intolerance. Co-administration of Tregitopesand islet cell antigens leads to the activation of natural T regulatorycells and the conversion of existing antigen specific effector T cell toa regulatory phenotype. In this way deleterious autoimmune response isredirected leading to the induction of antigen-specific adaptivetolerance. Modulation of auto-immune responses to autologous epitopes byinduction of antigen-specific tolerance can prevent ongoing beta-celldestruction. Accordingly, a Tregitope of the invention is useful inmethods for the prevention or treatment of diabetes.

Application to Hepatitus B (HBV) infection. Chronic HBV is usuallyeither acquired (by maternal fetal transmission) or can be a rareoutcome of acute HBV infection in adults. Acute exacerbations of chronichepatitis B (CH-B) are accompanied by increased cytotoxic T cellresponses to hepatitis B core and e antigens (HBcAg/HBeAg). In a recentstudy, the SYFPEITHI T cell epitope mapping system was used to predictMHC class II-restricted epitope peptides from the HBcAg and HbeAg. MHCclass II tetramers using the high scoring peptides were constructed andused to measure T_(Reg) and CTL frequencies. The results showed thatT_(Reg) cells specific for HBcAg declined during exacerbationsaccompanied by an increase in HBcAg peptide-specific cytotoxic T cells.During the tolerance phase, FOXp3-expressing T_(Reg) cell clones wereidentified. These data suggest that the decline of HbcAg T_(Reg) T cellsaccounts for the spontaneous exacerbations on the natural history ofchronic hepatitis B virus infection. Accordingly, a Tregitope of theinvention is useful in methods for the prevention or treatment of viralinfection, e.g., HBV infection.

Application to SLE. A TReg epitope that plays a role in Systemic LupusErythematosis (SLE) or Sjögren's syndrome has been defined. This peptideencompasses residues 131-151 (RIHMVYSKRSGKPRGYAFIEY; SEQ ID NO:67) ofthe spliceosome protein. Binding assays with soluble HLA class IImolecules and molecular modeling experiments indicated that the epitopebehaves as promiscuous epitope and binds to a large panel of human DRmolecules. In contrast to normal T cells and T cells from non-lupusautoimmune patients, PBMCs from 40% of randomly selected lupus patientscontain T cells that proliferate in response to peptide 131-151.Alteration of the ligand modified the T cell response, suggesting thatseveral populations of T cells responding to this peptide exist, amongwhich may be TReg cells. T regulatory epitopes have also been defined inSjögren's syndrome. Accordingly, a Tregitope of the inventionco-administered in combination with the epitope from above is useful inmethods for the prevention or treatment of SLE.

Application to Graves' Disease. Graves' disease is an autoimmunedisorder that is characterized by antibodies to self-thyroid stimulatinghormone receptor (TSHR) leading to leading to hyperthyroidism, or anabnormally strong release of hormones from the thyroid gland. Severalgenetic factors can influence susceptibility to Graves' disease. Femalesare much more likely to contract the disease than males; White and Asianpopulations are at higher risk than black populations and HLA DRB1-0301is closely associated with the disease. Accordingly, co-administrationof Tregitope(s) of the invention with TSHR or other Graves' diseaseantigens or portions thereof is useful in methods for the prevention ortreatment of Graves' disease.

Application to Autoimmune Thyroiditis. Autoimmune Thyroiditis is acondition that occurs when antibodies arise to self thyroid peroxidaseand/or thyroglobulin, which cause the gradual destruction of folliclesin the thyroid gland. HLA DR5 is closely associated with the disease.Accordingly, co-administration of Tregitope of the invention withthyroid peroxidase and/or thyroglobulin TSHR or portions thereof areuseful in methods for the prevention or treatment of autoimmunethyroiditis.

Application to the Design of Vaccine Vectors. A monoclonal antibodytargeting the dendritic cell surface receptor DEC-205 has shown promiseas a vaccine vector capable of targeting vaccine antigens to dendriticcells. The success of anti-DEC-205 as a stimulator of stronginflammatory immune responses, however, depends on co-administration ofnon-specific dendritic cell maturation factors. In their absence,anti-DEC-205 induces antigen-specific tolerance rather than immunity.Therefore, regulatory T-cell epitopes contained in anti-DEC-205 promotea tolerogenic reaction that is only overcome through theco-administration of non-specific immuno-stimulators. This point hasbeen verified experimentally, namely, that the Tregitopes contained inthe anti-DEC-205 vector cause antigen-specific expansion of regulatory Tcells and suppress inflammatory immune responses. Modifying thoseTregitopes such that they no longer bind to MHC molecules willsignificantly diminish tolerogenicity, enabling use of anti-DEC-205 asan effective stand alone antigen delivery system that obviates thedangers associated with non-specific activation of the immune system.

Kits

The methods described herein can be performed, e.g., by utilizingpre-packaged kits comprising at least one Tregitope composition of theinvention, which can be conveniently used, e.g., in clinical settings totreat subjects exhibiting symptoms or family history of a medicalcondition described herein. In one embodiment, the kit further comprisesinstructions for use of the at least one Tregitope composition of theinvention to treat subjects exhibiting symptoms or family history of amedical condition described herein.

Ex Vivo Expansion of T-Regulatory Cells Using Tregitopes

In another aspect, the invention provides ex vivo methods for theexpansion of regulatory T-cells. In one embodiment, the inventionprovides a method of expanding regulatory T-cells in a biologicalsample, the method comprising: (a) providing a biological sample from asubject; (b) isolating regulatory T-cells from the biological sample;and contacting the isolated regulatory T-cells with an effective amountof a Tregitope composition of the invention under conditions wherein theT-regulatory cells increase in number to yield an expanded regulatoryT-cell composition, thereby expanding the regulatory T-cells in thebiological sample. In one embodiment, the method further comprises thestep of administration of the expanded regulatory T-cell composition toa subject. In one embodiment, the subject administered the expandedregulatory T-cell composition is the same individual from which theoriginal biological sample was obtained, e.g., by autologoustransplantation of the expanded regulatory T-cell composition(Ruitenberg, J. et al., BMC Immunol., 7:11, 2006).

In Vitro Uses of Tregitope Compositions

In another aspect, the invention provides the use of the Tregitopecompositions of the invention as reagents in the study of regulatoryT-cell function in in vitro experimental models. In one embodiment, theinvention provides in vitro methods for stimulation of regulatoryT-cells in a biological sample, the method comprising: (a) providing abiological sample from a subject; (b) isolating regulatory T-cells fromthe biological sample; and contacting the isolated regulatory T-cellswith an effective amount of a Tregitope composition of the inventionunder conditions wherein the T-regulatory cells are stimulated to alterone or more biological function, thereby stimulating the regulatoryT-cells in the biological sample. In one embodiment, the inventionprovides in vitro methods for the measurement of binding Tregitope to aregulatory T-cells or fragment thereof.

The examples that follow are not to be construed as limiting the scopeof the invention in any manner. In light of the present disclosure,numerous embodiments within the scope of the claims will be apparent tothose of ordinary skill in the art.

EXEMPLIFICATION

Tregitopes were (1) identified using the T cell epitope mappingalgorithm EpiMatrix, (2) confirmed to bind to soluble HLA, (3) proven toengage natural regulatory T cells, (4) proven to suppress the immuneresponse to co-delivered antigens ex vivo (in human PBMC) and (3) provento suppress the immune response to co-delivered antigens in vivo (inmice). The methods for these discoveries are outlined below followed bythe corresponding results.

(1) Methods for the Identification of T Cell Epitopes and T Cell EpitopeClusters

T cells specifically recognize epitopes presented by antigen presentingcells (APCs) in the context of MHC (Major Histocompatibility Complex)Class II molecules. These T-helper epitopes can be represented as linearsequences comprising 7 to 30 contiguous amino acids that fit into theMHC Class II binding groove. A number of computer algorithms have beendeveloped and used for detecting Class II epitopes within proteinmolecules of various origins (De Groot, A. et al., AIDS Res. Hum.Retroviruses, 13: 539-541, 1997; Schafer, J. et al., Vaccine,16:1880-1884, 1998; De Groot, A. et al., Vaccine, 19:4385-95, 2001; DeGroot, A. et al., Vaccine, 21:4486-504, 2003). These “in silico”predictions of T-helper epitopes have been successfully applied to thedesign of vaccines and the deimmunization of therapeutic proteins.

The EpiMatrix system is a tool for predicting class I and class IIepitopes. The algorithm uses matrices for prediction of 9- and 10-merpeptides binding to HLA molecules. Each matrix is based onposition-specific coefficients related to amino acid binding affinitiesthat are elucidated by a method similar to, but not identical to, thepocket profile method (Sturniolo, T. et al., Nat. Biotechnol.,17:555-561, 1999). The EpiMatrix system has been used to prospectivelypredict a large number of epitopes that have been confirmed in vitro andin vivo. The entire amino acid of any given sequence is first parsedinto overlapping 9-mer frames where each frame overlaps the last byeight amino acids. Each frame is then scored for predicted affinity toeach of eight common Class II HLA haplotypes (DRB1*0101, DRB1*0301,DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501).Due to their prevalence and their difference from each other, theseeight alleles cover around 97% of human populations worldwide. EpiMatrixraw scores are then normalized with respect to a score distributionderived from a very large set of randomly generated peptide sequences.The resulting “Z” scores are normally distributed and directlycomparable across alleles.

EpiMatrix peptide scoring. It was determined that any peptide scoringabove 1.64 on the EpiMatrix “Z” scale (approximately the top 5% of anygiven peptide set) has a significant chance of binding to the MHCmolecule for which it was predicted. Peptides scoring above 2.32 on thescale (the top 1%) are extremely likely to bind; most published T cellepitopes fall within this range of scores. Previous studies have alsodemonstrated that EpiMatrix accurately predicts published MHC ligandsand T cell epitopes.

Identification of promiscuous T cell Epitope Clusters. Following epitopemapping, the result set produced by the EpiMatrix algorithm is screenedfor the presence of T cell epitope clusters and EpiBars. Potential Tcell epitopes are not randomly distributed throughout protein sequencesbut instead tend to “cluster.” T cell epitope “clusters” range from 9 toroughly 30 amino acids in length and, considering their affinity tomultiple alleles and across multiple frames, contain anywhere from 4 to40 binding motifs. Using a proprietary algorithm know as ClustiMer,putative T cell epitope clusters are identified. Briefly, the EpiMatrixscores of each 9-mer peptide analyzed are aggregated and checked againsta statistically derived threshold value. High scoring 9-mers are thenextended one amino acid at a time. The scores of the extended sequencesare then re-aggregated and compared to a revised threshold value. Theprocess is repeated until the proposed extension no longer improves theoverall score of the cluster. Tregitope(s) identified in the presentstudies were identified by the ClustiMer algorithm as T cell epitopeclusters. They contain significant numbers of putative T cell epitopesand EpiBars indicating a high potential for MHC binding and T cellreactivity.

(2) Methods for the Assessment of Peptide Synthesis and Binding toSoluble MHC.

Synthesis of peptides. Tregitopes can be produced by direct chemicalsynthesis or by recombinant methods (Sambrook et al., Molecular Cloning:A Laboratory Manual, 2 ed., Cold Spring Harbor Laboratory Press,(1989)). Peptides corresponding to the Tregitopes of the invention wereprepared by 9-fluoronylmethoxycarbonyl (Fmoc) synthesis at New Englandpeptide and on an automated Rainen Symphony/Protein Technologiessynthesizer (Synpep, Dublin, Calif.). The peptides were delivered >80%pure as ascertained by HPLC, mass spectrometry and UV scan (ensuringpurity, mass and spectrum, respectively).

Binding of produced peptides. Non-biotinylated test peptide is suspendedin a 96-well polypropylene plate in final concentrations ranging from0.1 μM-400 μM in triplicate wells. Purified recombinant HLA Class IImolecules in a solution containing 1 mM PefaBloc, 0.75%n-octyl-B-D-glucopyranoside in 150 mM citrate-phosphate buffer (pH 5.4),were then added to these wells at a final concentration of 200 ng/well.The 96-well plates are incubated at 37° C. in 6% CO₂ for 45 minutes.Following the incubation, biotinylated Flu HA peptide 307-319 (oranother suitable control peptide) is added to a final concentration of0.1 μM per well and incubated at 37° C. for 20 hours. The contents ofeach well are then added to a 96-well high binding ELISA platepreviously coated with the anti-human HLA-DR L243 capture antibody(Becton Dickenson) and incubated at 4° C. for 20 hours. The plate wasthen developed by the addition of 100 μl (10 μg/ml) of Europium-labeledStreptavidin (Perkin-Elmer) and 100 μl Enhancement Buffer (Perkin-Elmer)to each well. The reaction was incubated in the dark at room temperaturefor 15-30 minutes and then fluorescence was measured on a Wallac Victor3-V time-resolved fluourometer. IC₅₀ values were then calculated bynon-linear regression analysis using the SigmaPlot analysis program.Based on comparisons with known peptides, an IC₅₀ of 250 μM or more isindicative of weak binding and an IC₅₀ of 400 μM or more is indicativeof a non-binding interaction.

(3) Methods for Assessing the Ability of Peptides to Engage NaturalRegulatory T Cells.

T-cell isolation. This research program involves donated blood obtainedfrom the Rhode Island Blood Bank in Providence, blood from volunteersrecruited at Clinical Partners, Johnston, R.I., blood obtained fromvolunteers recruited by Stallergenes, Paris, France, and samplesobtained from a commercial supplier. Donor blood was obtained inaccordance with all federal guidelines and in accordance withStallergenes and EpiVax institutional policies. The protocol forobtaining donor blood was approved by the respective institutionalreview boards. Peripheral blood mononuclear cells (PBMC) were isolatedaccording to the Accuspin protocol (Sigma-Aldrich, St. Louis, Mo.).Cryopreserved PBMC from dust-mite-allergic individuals were obtainedfrom Cellular Technologies Ltd. (Cleveland, Ohio).

Natural T reg assay. Human PBMCS are stimulated directly ex vivo for 4days in the presence of tetanus toxin peptide TT₈₃₀₋₈₄₄ alone, Tregitopealone, phytohemagglutinin alone (a mitogenic positive control) or nostimulus. 1×10⁶ cells were stained with anti-CD4-FITC (clone RPA-T4;eBioscience) and anti-CD25-APC (clone BC96; eBioscience) antibodies for30 minutes on ice in Flow Staining Buffer (eBioscience) and washed twicewith buffer. Following cell surface staining, cells are fixed andpermeabilized (eBioscience) and stained intracellulary for FOXp3 (clonePCH101; eBioscience) following manufacturer's protocol. The frequency ofFOXp3 positive CD4+/CD25+ T cells under various culture conditions isenumerated by the Flowjo analysis software. T cell activation isindicated by increases in CD4+CD25+ expression, which, when accompaniedby an increase in FOXp3 expression, is indicative that the activatedcells are regulatory.

(4) Methods for Assessing the Ability of Peptides to Suppress theResponse to Co-Administered Antigens Ex Vivo.

Bystander suppression assay. Isolated PBMCs were cultured for 8 days at37° C. 5% CO₂ in presence of either an immunogenic antigen alone or thatantigen in the presence of Tregitope peptide. Test antigens were addedat 10 μg/ml and include 1) classic antigens such as, for example,tetanus toxin peptide TT₈₃₀₋₈₄₄, influenza hemagglutinin peptide307-319, vaccinia peptide epitopes and the CEF positive control peptidepool (NIH AIDS Research & Reference Reagent Program at the websiteaidsreagent.org; Currier, J. et al., J. Immunol. Methods, 260:157-72,2002; Mwau, M. et al., AIDS Res. Hum. Retroviruses, 18:611-8, 2002), 2)protein therapeutics such as Botulinum Neurotoxin A, autologousautoantigens such as Thyroid Hormone Stimulating hormone and complementcomponent C3d. Test antigens also included allergens: birch tree pollenantigen Betv1, House dust mite lysate and the purified house dust miteantigen, Der P2. Recombinant IL-2 (10 IU/ml) and IL-7 (20 ng/ml) wereadded to PBMC cultures on day 2. After 8 days of stimulation, cells wereharvested and washed several times with PBS and assayed according to thehuman cytokine release assays described below.

Human IFN-γELISpot. IFN-γ ELISpot assays are performed using Human IFN-γELISpot kits purchased from Mabtech. Target peptides are added at 10μg/ml to triplicate wells containing 250,000 human peripheral bloodmononuclear cells in RPM11640 with 10% human serum and incubated foreighteen to twenty-two hours at 37° C. under a 5% CO₂ atmosphere.Triplicate wells are plated with PHA at 10 μg/mL. Six wells with nopeptide are used for background determination. A response is consideredpositive if the number of spots in the peptide test wells isstatistically different (p<0.05) from that of the control wells by theMann-Whitney U test. In general, responses are considered positive ifthe number of spots is at least four times background and greater than50 spots per one million cells over background (1 response overbackground per 20,000 splenocytes). Results are recorded as the averagenumber of spots over background and adjusted to spots per one millioncells seeded. Suppression rates of 10% or greater, when determined to bestatistically significant, are considered statistically significant.

Human IFN-γELISpot. IFN-γ ELISpot assays are performed using Human IL-4ELISpot kits purchased from Mabtech. Target peptides are added at 10μg/ml to triplicate wells containing 250,000 human peripheral bloodmononuclear cells in RPM11640 with 10% human serum and incubated foreighteen to twenty-two hours at 37° C. under a 5% CO₂ atmosphere.Triplicate wells are plated with PHA at 10 μg/mL. Six wells with nopeptide are used for background determination. A response is consideredpositive if the number of spots in the peptide test wells isstatistically different (p<0.05) from that of the control wells by theMann-Whitney U test. In general, responses are considered positive ifthe number of spots is at least four times background and greater than50 spots per one million cells over background (1 response overbackground per 20,000 splenocytes). Results are recorded as the averagenumber of spots over background and adjusted to spots per one millioncells seeded. Suppression rates of 10% or greater, when determined to bestatistically significant, are considered statistically significant.

Human IL-4 ELISpot. IL-4 ELISpot assays are performed using Human IL-4ELISpot kits purchased from Mabtech. Target peptides are added at 10μg/ml to triplicate wells containing 250,000 human peripheral bloodmononuclear cells in RPMI1640 with 10% human serum and incubated foreighteen to twenty-two hours at 37° C. under a 5% CO₂ atmosphere.Triplicate wells are plated with PHA at 2 μg/mL. Six wells with nopeptide are used for background determination. Statistical tests werecarried out using a variant permutation test (Hudgens, M. et al., J.Immunol. Methods, 288:19-34, 2004). A response is considered positive ifthe number of spots in the peptide test wells is statistically different(p<0.01) from that of the control wells. In general, responses areconsidered positive if the number of spots is at least four timesbackground and greater than 50 spots per one million cells overbackground (1 response over background per 20,000 splenocytes). Resultsare recorded as the average number of spots over background and adjustedto spots per one million cells seeded. Suppression rates of 10% orgreater, when determined to be statistically significant, are consideredsignificant.

Human IFN-γELISA. Target peptides are added at 10 μg/ml to culturescontaining human peripheral blood mononuclear cells in RPM11640 with 10%human serum and incubated for eighteen to twenty-two hours at 37° C.under a 5% CO₂ atmosphere. Cultures stimulated with PHA at 10 μg/mL orwith no peptide are used as controls. Human IFN-γ quantitative sandwichELISAs were performed using R&D Systems Quantikine ELISA kits. Apolyclonal antibody specific for IFN-γ is pre-coated onto a 96-wellmicrotiter plate. Kit-provided standards and cell supernatant samplesincluding PHA and no-peptide controls (100 μl) are pipetted into thewells and any IFN-γ present is bound by the immobilized antibody over 2hours at room temperature. After washing away unbound substances, anenzyme-linked polyclonal antibody specific for IFN-γ is added to thewells for a two hour incubation at room temperature. Following a wash toremove any unbound antibody-enzyme reagent, a substrate solution isadded to the wells for 30 minutes and color developed in proportion tothe amount of bound IFN-γ. The color development is stopped and theintensity of the color at 450 nm measured on a Wallac Victor3.Correction for optical imperfections in the plate is made by subtractionof intensities at 540 nm from the 450 nm values. Differences in cytokinelevels between experimental groups were evaluated by t-test. A responseis considered positive if the observed difference in cytokine expressionbetween the experimental and control wells is statistically different(p<0.01).

Multiplexed human cytokine/chemokine ELISA. Supernatants from PBMCcultures are evaluated for cytokines and chemokine levels using theSearchLight multiplex ELISA technology. Human cytokines that aremeasured include IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40,IL-12p70, TNFα and TGFβ. Human chemokines that are measured includeMCP-1, MIP-1α and MIP-1β. SearchLight□ Proteomic Arrays are aquantitative multiplexed sandwich ELISA containing up to 16 differentcapture antibodies spotted on the bottom of a 96-well polystyrenemicrotiter plate. Each antibody captures specific protein detected witha biotinylated antibody, followed by the addition of streptavidin-HRPand lastly, SuperSignal ELISA Femto Chemiluminescent substrate detectedwith a charge-coupled device (CCD) camera. Differences in cytokinelevels between experimental groups were evaluated by t-test. A responseis considered positive if the observed difference in cytokine expressionbetween the experimental and control wells is statistically different(p<0.01).

Cell separations/depletions methods. Human Treg cell populations aredepleted or positively isolated from PBMC using the invitrogen dynabeadssystem (for human CD4 and CD25) according to manufacturer's instructions(InVitrogen, Carlsbad, Calif.).

(5) Methods for the Suppression of Response to Co-Administered AntigensIn Vivo.

To measure the immunosuppressive effects of Tregitopes onprotein-induced effector responses in a living system, experiments areperformed using a murine model. Groups of mice are immunized with anantigen alone, a cocktail of antigen and tregitope, or with an antigenfused to tregitope. A negative control group (solvent alone) is alsoassessed. One week following the final injection, the mice aresacrificed in accordance with all institutional and federal guidelinesand spleens harvested. Freshly isolated mouse splenocytes are used toassay the cellular immune response in vivo. Single splenocytesuspensions are prepared and used in the assays below. Whole blood isalso obtained by cardiac puncture and serum collected for use inquantifying antibody response to the co-administered antigen.

Murine IFN-γELISpot. IFN-γ ELISpot assays are performed using murineIFN-γ ELISpot kits purchased from Mabtech. Target peptides are added at10 μg/ml to tripliocate wells containing 300,000 murine splenocytes (inRPM11640 with 10% FCS) and incubated for eighteen to twenty-two hours at37° C. under a 5% CO₂ atmosphere. Triplicate wells are plated with ConAat 10 μg/mL. Six wells with no peptide are used for backgrounddetermination. A response is considered positive if the number of spotsin the peptide test wells is statistically different (p<0.05) from thatof the control wells by the Mann-Whitney U test. In general, responsesare considered positive if the number of spots is at least four timesbackground and greater than 50 spots per one million cells overbackground (1 response over background per 20,000 splenocytes). Resultsare recorded as the average number of spots over background and adjustedto spots per one million cells seeded.

Murine IL-4 ELISpot. IL-4 ELISpot assays are performed using murine IL-4ELISpot kits purchased from Mabtech. Target peptides are added at 10μg/ml to triplicate wells containing 300,000 murine splenocytes (inRPM11640 with 10% FCS) and incubated for eighteen to twenty-two hours at37° C. under a 5% CO₂ atmosphere. Triplicate wells are plated with ConAat 10 μg/mL. Six wells with no peptide are used for backgrounddetermination. Statistical tests were carried out using a variantpermutation test (Hudgens, M. et al., J. Immunol. Methods, 288:19-34,2004). A response is considered positive if the number of spots in thepeptide test wells is statistically different (p<0.01) from that of thecontrol wells. In general, responses are considered positive if thenumber of spots is at least four times background and greater than 50spots per one million cells over background (1 response over backgroundper 20,000 splenocytes). Results are recorded as the average number ofspots over background and adjusted to spots per one million cellsseeded.

Murine IFN-γELISA. Target peptides are added at 10 μg/ml to culturescontaining human peripheral blood mononuclear cells in RPM11640 with 10%human serum and incubated for eighteen to twenty-two hours at 37° C.under a 5% CO₂ atmosphere. Cultures stimulated with PHA at 10 μg/mL orwith no peptide are used as controls. Mouse IFN-γ quantitative sandwichELISAs were performed using R&D Systems Quantikine ELISA kits. Apolyclonal antibody specific for IFN-γ is pre-coated onto a 96-wellmicrotiter plate. Kit-provided standards and cell supernatant samplesincluding PHA and no-peptide controls (100 μl) are pipetted into thewells and any IFN-γ □present is bound by the immobilized antibody overtwo hours at room temperature. After washing away unbound substances, anenzyme-linked polyclonal antibody specific for IFN-γ is added to thewells for a two hour incubation at room temperature. Following a wash toremove any unbound antibody-enzyme reagent, a substrate solution isadded to the wells for 30 minutes and color developed in proportion tothe amount of bound IFN-γ. The color development is stopped and theintensity of the color at 450 nm measured on a Wallac Victor3.Correction for optical imperfections in the plate is made by subtractionof intensities at 540 nm from the 450 nm values. Differences in cytokinelevels between experimental groups were evaluated by t-test. A responseis considered positive if the observed difference in cytokine expressionbetween the experimental and control wells is statistically different(p<0.01).

Flow Cytometry. Splenocytes are plated in 96-well tissue culture platesat 2×10⁶ cells/well in RPMI 1640 supplemented with 10% FCS, 100 U/mLpenicillin, 100 μg/mL streptomycin sulfate. An unstimulated and positivecontrol (ConA) are included in each assay. Cells are incubated overnightat 37° C. at 5% CO₂. Following incubation, the cells are washed in PBScontaining 1% bovine serum albumin and stained with surface antibodies(e.g., CD4, CD25). Cells are then washed and fixed using theCytofix/Cytoperm kit (BD PharMingen) according to manufacturer'sinstructions. Following fixation, the cells are washed twice in Cytopermbuffer and stained with antibodies against intracellular markers (e.g.,FOXp3, IL-10). Following staining, the cells are washed and fixed withPBS containing 1% paraformaldehyde in preparation for flow cytometry.Cells are analyzed on a BD Facscalibur machine. 20,000 events arecollected per sample. Data analysis is performed using FloJo software.All data are background-subtracted. Comparisons between groups are basedon a Wilcoxon rank sum test. A significance of p<0.05 is applied forpairwise comparisons and p<0.01 is used for multiple comparisons.

Cell separations/depletions methods. Murine Treg cell populations aredepleted or positively isolated from PBMC using the InVitrogen dynabeadssystem (for murine CD4 and CD25) according to manufacturer'sinstructions (InVitrogen, Carlsbad, Calif.).

Quantification of antibodies against co administered antigen.Quantification of IgG antibody to antigens was determined byantibody-capture ELISA. Antigen (10 μg/mL) is dissolved in carbonatebuffer and placed into a 96-well microtiter plate overnight at 4° C. Theplates were then washed with phosphate-buffered saline containing 0.05%Tween 20 (PBST) and blocked for three hours at room temperature with 5%fetal bovine serum (FBS; Gibco) in PBS. Serial dilutions of sera in 0.5%FBS/PBS are added to the plates and incubated at room temperature fortwo hours. The microtiter plates are then washed with PBST and 100 μLgoat anti-mouse IgG (gamma-chain specific) conjugated to horseradishperoxidase (Southern Biotechnology Associates) diluted 1:10000 in 0.5%FBS/PBS is added to each well. Microtiter plates are washed in PBST andthen developed with 3,3′,5,5′-tetramethylbenzidine (TMB; Moss).Absorbances were read at a wavelength of 450 nm measured on a WallacVictor3. Correction for optical imperfections in the plate is made bysubtraction of intensities at 540 nm from the 450 nm values.

Example 1. Identification of a Tregitope Composition

Identification of epitopes in Human IgG Proteins as regulatory. Afterevaluating a large number of antibodies for immunogenic potential, arecurring pattern was observed. Certain epitope clusters were occurringin multiple antibodies. Not wishing to be bound by theory, it wasreasoned that highly conserved epitope clusters were unlikely to bepromoting anti-antibody immune responses. It was further reasoned thatthese recurring patterns might be either passively tolerated by theimmune system or actively engaging regulatory T cells responsible forsuppressing anti-antibody immune response. Comparing the sequences ofthe recurring epitope clusters to the protein database at GenBankestablished 19 regions contained in the sequences of IgG antibodies thatwere both conserved and potentially capable of stimulating regulatory Tcells (See FIG. 18A and FIG. 18B).

As shown in FIG. 18A and FIG. 18B, according to the EpiMatrix system,all 19 of these regions have significant immune stimulatory potential,each one containing at least one and at most 14 binding motifs andscoring between one and 25 on the EpiVax immunogenicity scale. Inaddition several of these sequences contained one or more “EpiBars”.EpiBars are single 9 mer frames that are expected to bind to at least 4different Class II HLA. EpiBars are a marker for increasedimmuno-stimulatory potential.

Conservation. All the IgG derived putative Tregitope sequences werecompared to the germline sequences of IgG1, IgG2, IgG3, IgG4, IgA, IgE,IgD and IgM through visual inspection. The IgG-derived Tregitopes werefound to be highly conserved in the germline sequences of IgG1, IgG2,IgG3 and IgG4. No homology was found in the germline sequences of IgA,IgE, IgD or IgM. The sequences of the additional Tregitopes are alsohighly conserved among (variants of human proteins) and are generallypresent in the circulation in large amounts.

Species. Homology analysis of the IgG-derived Tregitopes to non-humanspecies was performed. The sequences were uploaded into the Basic LocalAlignment Search Tool (BLAST) via the NCBI website(ncbi.nlm.nih.gov/blast). The BLAST program compares protein sequencesto sequence databases and calculates the statistical significance ofmatches in order to find regions of local similarity between sequences.The IgG-derived Tregitopes were found to be conserved across non-humanspecies such as mouse, rat, cat, camel, cow and non-human primates. FIG.19 illustrates a BLAST report of Tregitope-289 (SEQ NO: 4).

Identification of regulatory epitopes in common circulating humanproteins. In a subsequent analysis EpiVax identified a set of common andcirculating proteins that might also contain Tregitopes. The analyzedprotein set included isolates of human: Actin, Albumin, Collagen,Fibrinogen, Haptoglobin, Keratin, Myosin, Osteocalcin, Prostaglandin,Superoxide Dismutase, Titin and Transferrin. Common isolates of eachprotein were analyzed via EpiMatrix and ClustiMer as described above anda set of high scoring and highly conserved putative T cell epitopeclusters was selected for further analysis. See FIG. 18A and FIG. 18B,SEQ ID NOS:38-58.

Example 2. Synthesis and Characterization of a Tregitope Composition byBinding to HLA Class II Molecules

Soluble MHC binding assays were performed on the synthetic IgGTregitopes according to the methods described above. IC₅₀ values (μM)were derived by a six point inhibition curve of a strong binding controlpeptide. As depicted in FIG. 20, the Tregitopes identified by in silicoanalysis bound to human MHC molecules.

Additional assays related to structural modifications to amino andcarboxy termini. Modifications to the amino and caboxy termini ofpeptides have been shown to alter MHC binding, proteolytic degradationand T cell activation (Maillère, B. et al., Mol. Immunol., 32:1377-85,1995; Allen, P. et al., Int. Immunol., 1:141-50, 1989). If the observedactivation of nTregs were indeed due to Tregitope-specific TCRrecognition, then fine alterations at the carboxy terminus of theTregitope peptide should lead to differential suppressive effects. Thesame Tregitope peptide sequence was synthesized with and without aC-terminal amide cap. The uncapped peptide was evaluated for affinity toDRB1*0101 and DRB1*1501 in HLA binding assays and shown to bind to bothalleles with higher affinity than did the capped peptide. Using PBMCfrom a DRB1*0101 subject, the ability of Tregitope peptides (capped anduncapped) to suppress responses to co-incubated CEF, a MHC class Iimmunogenic peptide pool, was then investigated. The cells werestimulated on day 1 and cultured for 6 days. On day 7 the cells werecollected and half were stained for CD4, CD25 and CD127 and analyzed byflow cytometry. The remaining cells were added to an IFN-γ ELISpot plateand re-stimulated with CEF. The co-cultures with the C-terminalamide-capped Tregitope led to an increase in CD4+CD25+CD127low Tregscompared to the uncapped Tregitope-289 (FIG. 2, left panel). Consistentwith previous studies that have shown that CD4+CD25+CD127low Tregs arehighly suppressive, the capped Tregiotpe-289, but not the uncappedTregitope-289, was able to suppress CEF-specific IFN-γ secretion (FIG.2, right panel).

Subsequent analysis showed a small 1 dalton change between the cappedand uncapped versions of Tregitope-289 (SEQ NO: 4). Tregitope-289amidated peptide is 1 dalton smaller by mass spectrometry analysis.Amidation of the C-terminus of Tregitope-289 is herein demonstrated toalter its binding and functional characteristics. Because the cappedversion of Tregitope-289 peptide demonstrated better functionality, thecapped (amidated) peptide was used in all subsequent assays. In furthersupport, results displayed here for Tregitope-289 refer to the cappedversion. Both capped and uncapped versions of the Tregitopes describedherein are encompassed by the present invention.

Example 3. Characterization of a Tregitope Composition by Stimulation ofNatural Regulatory T Cells

Human PBMCS were stimulated directly ex vivo for 4 days in the presenceof tetanus toxin peptide TT₈₃₀₋₈₄₄ alone, Tregitope-289 alone,phytohemagglutinin (a mitogenic positive control) alone, or no stimulus.1×10⁶ cells were stained with anti-CD4-FITC (clone RPA-T4; eBioscience)and anti-CD25-APC (clone BC96; eBioscience) antibodies for 30 minutes onice in Flow Staining Buffer (eBioscience) and washed twice with buffer.Following cell-surface staining, cells were fixed and permeabilized(eBioscience) and stained intracellulary for Foxp3 (clone PCH101;eBioscience) following manufacturer's protocol. The frequency of FoxP3positive CD4+/CD25+ T cells under various culture conditions wasenumerated by Flowjo analysis software. There were similar increases inCD25 expression in both the Tetanus- and Tregitope-stimulated samplesindicating T cell activation by both peptides (FIG. 3; results shown forTregitope-289). Expression of FoxP3 within the CD4+CD25+ subset,however, differed significantly depending on the stimulus used. Tetanusstimulation led to a 7% decrease in expression of FoxP3, whereasTregitope stimulation led to a more than two-fold increase inexpression, indicating Th and nTreg activation, respectively.

Example 4. Characterization of a Tregitope Composition by Suppression ofCo-Administered Antigen In Vitro

4A: Tregitope-167 and Tregitope-134 Down-Regulate Effector Responses andUpregulate Regulatory Responses to Coadministered Antigens In Vitro.

PBMCs were cultured for 8 days with either a) pool of immunogenicpeptides alone, b) a pool of immunogenic peptides with hTregitope-167,or c) a pool of immunogenic peptides with hTregitope-134. Cells wereharvested and washed with PBS Cells (2×10⁵ cells/well) were plated into96-well plate and re-stimulated with the immunogenic peptide pool alone,the immunogenic peptide pool and Tregitope, or no peptide (negativecontrol) for 65 hours. Supernatants were analyzed by multiplexed ELISAanalysis as described above. The co-incubation of Tregitope during theinitial stimulation led to an increase in secretion of the regulatorycytokines and chemokines, IL-10 and MCP-1 and a decrease in thesecretion of helper T cell cytokines and chemokines, IL-5, IL-6, IFN-γand MIP-1α □ demonstrating the ability of Tregitopes to engage andactivate regulatory T cells (FIG. 4).

4B: Tregitope-289 Downregulates Effector Responses and UpregulatesRegulatory Responses to Co-Administered Antigen In Vitro.

PBMCs were cultured for 8 days with either a) pool of immunogenicpeptides alone, b) a pool of immunogenic peptides with Tregitope-289, orb) a pool of immunogenic peptides withTregitope-289. The peptides in theimmunogenic peptide pool were derived from C3d, an immunogenicautologous protein (Knopf, P. et al., Immunol. Cell Biol., 2008 Jan. 8;doi: 10.1038/sj.icb.7100147). Cells were harvested and washed with PBS.As described, Cells (2×10⁵ cells/well) were plated into 96-well plateand re-stimulated in triplicate wells with each condition: C3d poolalone, C3d pool+Tregitope, PHA control, or no peptide (negative control)for 65 hours. Supernatants were analyzed by multiplexed ELISA analysis.Response to positive control PHA was robust following both cultureconditions. The co-incubation of Tregitope during the initialstimulation led to an increase in secretion of the regulatory cytokineIL-10, a slight increase in the regulatory chemokine TGFβ, and adecrease in the secretion of the helper T cell cytokines and chemokinesIFNγ and MIP 1α further demonstrating the ability of Tregitopes toengage and activate regulatory T cells (FIG. 5).

4C: A Pool of Tregitopes Downregulates Effector Auto-Immune Responses toCo-Administered Antigen In Vitro.

Co-incubation with epitopes derived from TSHR, the target antigen ofGraves' disease, suppresses immune response to the epitopes in PBMC froma patient with Graves' disease. PBMCs were cultured for 8 days with TSHRpeptide pools (pool) with or without a pool of Tregitope peptides(Tregitope-134, Tregitope-167, Tregitope-289). Cells were harvested andwashed with PBS. As described above, 2.5×10⁵ cells were re-stimulated inan IL-4 ELISpot plate with either 1) individual TSHR epitopes+the poolof Tregitope-134, Tregitope-167, Tregitope-289), 2) a pool of TSHRepitopes+the pool of Tregitope-134, Tregitope-167, Tregitope-289 or 3)no stimulus control. Response to positive control PHA was robustfollowing both culture conditions.

The co-incubation of antigen (TSHR peptides) with Tregitope duringre-stimulation led to a significant decrease in IL-4 spot-forming cells.This data shows that Tregitopes suppress the cytokine secretion ofeffector T cells (FIG. 6).

4D: Individual Tregitopes Downregulate Effector Responses to CEF, a Poolof Immunodominant Co-Administered Peptide Antigens In Vitro.

Co-incubation with Tregitope suppresses immune response to CEF, a poolof Immunodominant peptide epitopes derived from common pathogens. PBMCswere cultured for 8 days with or without individual Tregitope peptides:Tregitope-289, Tregitope 294, Tregitope-029, Tregitope-074,Tregitope-009. Cells were harvested and washed with PBS. As describedabove, 2.5×10⁵ cells were re-stimulated in an IFN-γ ELISpot plate witheither CEF alone, PHA positive control (not shown) or no-stimuluscontrol. Response to positive control PHA was robust following bothculture conditions.

The co-incubation of antigen (CEF) with Tregitope during incubation ledto a significant decrease in IFN-γ spot-forming cells in response torestimulation with CEF. These data show that Tregitopes suppress thecytokine secretion of effector T cells (FIG. 7) 4E: A pool of Tregitopesdownregulates in vitro effector response to co-administered therapeuticprotein antigen.

Co-incubation with Tregitope suppresses immune response to peptideepitopes derived from Botulinum neurotoxin, a protein used to treatdystonia (movement disorders). PBMCs from a subject with evidence ofinhibitors (anti-BoNT antibodies) were cultured for 8 days with orwithout a pool of Tregitope peptides (Tregitope-167, Tregitope-134,Tregitope-289). Cells were harvested and washed with PBS. As describedabove, 2.5×10⁵ cells were re-stimulated in an IFN-γ ELISpot plate withindividual BoNT peptides, a pool of BoNT peptides, PHA positive control(not shown) or no-stimulus control. Peptides for which there was nosignificant baseline response are not shown. Response to positivecontrol PHA was robust following both culture conditions.

The co-incubation of antigen (CEF) with Tregitope during incubation ledto a significant decrease in IFN-γ spot-forming cells in response torestimulation with CEF. These data show that Tregitopes suppress thecytokine secretion of effector T cells in response to an immunogenictherapeutic protein (FIG. 8 and FIG. 21).

4F: Tregitope-289 and Tregitope-134 down-regulate proliferation inresponse to co-administered immunodominant antigens in vitro.

CEF is a commercially available pool of immunodominant peptide epitopesfrom common pathogens. PBMCs were cultured for 8 days with CEF alone,CEF+Tregitope-134, or CEF+Tregitope-289. Cells were harvested and washedwith PBS. 2×10⁶ cells were pre-labeled with CFSE dye (Invitrogen) bystandard protocol and re-stimulated for 65 hours with CEF pool, or nopeptide (negative control), or PHA mitogen control; supernatants werecollected and IFN-γ ELISAs were performed as described above. Responseto positive control PHA was robust following both culture conditions.The co-incubation of Tregitope during re-stimulation led to asignificant decrease in IFN-γ production (left panel), which correlatedwith the reduction in the proliferation of effector T cells (FIG. 9,right panel).

4G: Tregitope-289 Downregulates Proliferation in Response toCo-Administered Antigen In Vitro.

PBMCs from a subject previously immunized with vaccinia were culturedfor 8 days with either an immunogenic vaccinia peptide alone or animmunogenic vaccinia peptide with Tregitope-289 as described above.Cells were harvested and washed with PBS. 2×10⁶ cells were pre-labeledwith CFSE dye (Invitrogen) by standard protocol and re-stimulated withthe vaccinia peptide, vaccinia peptide and Tregitope-289, or no peptide(negative control) for 65 hours. The co-incubation of Tregitope duringincubation led to a significant decrease in proliferation of theeffector T cells further demonstrating the ability of regulatory T cellsactivated by Tregitope to reduce the proliferation of effector T cells(FIG. 10).

4H: Tregitope Suppression is Mediated by Cells with a RegulatoryPhenotype (CD4+CD25Hi T Cells) and Upregulation of IL-10.

Two samples of PBMC from a single dust-mite-allergic individual wereprepared. One sample was stained with anti-CD4 and anti-CD25 antibodiesand analyzed by flow cytometry. In this sample the CD4+CD25Hi subset ofcells were depleted from the remaining PBMC by the methods describedabove. The other sample was left intact. The two samples were thenco-stimulated HDM lysate with or without Tregitope-289.CD4+CD25Hi-depleted PBMC were less able to suppress IFN-γ than werenon-depleted PBMC, indicating that suppressive effects of Tregitopes aremediated by CD4+CD25Hi cells. In an ancillary analysis in (intact)PBMCs, CD4+ proliferative responses to HDM lysate were suppressedfollowing co-incubation with HDM lysate and Tregitope-289 as comparedwith incubation with HDM lysate alone.

FIG. 11 documents the requirement for CD4+/CD25hi T cells in the initialco-incubation. In the presence of CD4+CD25hi cells, co-stimulation withTregitope-289 and HDM caused suppression of gamma interferon releasefollowing restimulation with HDM alone; in the absence of CD4+CD25hicells (sorted prior to the incubation, co-stimulation with Tregitope-289and HDM was associated with a lower amount of suppression (16%: 16.5 to12.4 μg/ml) as compared with a higher amount of suppression (65%: 33.5to 11.8 μg/ml) following restimulation with HDM alone. FIG. 11 show thatthe cellular subset containing Tregs is necessary for the induction oftolerance to an antigen.

4I: Tregitope Co-Incubation Causes Expansion of Cells with a RegulatoryPhenotype (CD4+CD25Hi T Cells) and Upregulation of Regulatory CytokineIL-10 in Response to an Allergen.

Induction of adaptive tolerance: to determine if Tregitope nTregactivation could lead to generation of allergen-specific aTReg, PBMC(from dust mite allergic individuals) first incubated for 8 days withDust Mite (DM) antigen alone, dust mite antigen+Tregitope-289, or dustmite antigen+Tregitope-167 were analyzed. As shown in the top panel(FIG. 12), co-incubation of PBMC with DM antigen and Tregitope-289 ledto a nearly four-fold expansion of CD4+CD25Hi cells; the same was trueof PBMC co-incubated with DM antigen and Tregitope-167 (1.6 to 7.5%). Inboth Tregitope co-incubations, IL-10 secretion was also found to beincreased five-fold (FIG. 12, bottom panel); a finding consistent withthe possibility that the increased CD4+CD25Hi cells may be HDM-specificadaptive Treg. One of skill in the art can confirm that the expandedCD4+CD25hi population is secreting IL-10 in this in vitro assay. TheIL-10 secretion in response to the co-incubated antigen, in the presenceof an expanded population of CD4+CD25hi Tregulatory cells, indicatesthat adaptive Tregs were induced during the coincubation with antigen.

These data show, in the same patient and the same experiment, theexpansion of CD4 CD25hi T cells following co-incubation withTregitope-289 and DM antigen and following co-incubation withTregitope-167 and DM antigen; and the amount of IL-10 secreted by theco-incubated cells following restimulation with HDM alone.

4J: Tregitope Co-Incubation Causes Suppression of Antigen-SpecificAllergic Th2 Responses.

Tregitope co-incubation also led to a significant decrease in expressionof CCR4, CD30, CRTH2, and CCR6, which have been shown to be associatedwith Th2 responses. Modulation of cytokine responses byallergen-specific CD4+ T cells following extended Tregitopeco-stimulation was subsequently evaluated. After 30 days in culture,Tregitope co-stimulation contributed to the development of a mixedpopulation of Bet v 1₁₁₄₁₋₁₁₅₅-specific CD4+ T cells. Followingprolonged stimulation with antigen and Tregitope, 42% of theseepitope-specific cells were neither IL5 nor IFN-γ positive, and 44%demonstrated a shift to a Th-1-like increased interferon response inthis prolonged incubation (FIG. 13).

Of note, the study subjects were selected for presence of HLA DR*1 1501to improve the chances of tetramer binding; the effect of Tregitope-167was more pronounced (five fold increase in Treg induction) than forTregitope 289 (three fold increase). Tregitope-289 was not shown to bindto DR 1501 in HLB binding assays. In contrast Tregitope-167 binds avidlyto HLA 1501 (87% inhibition of binding at 50 μM).

Example 5. Characterization of a Tregitope Composition by Suppression ofCo-Administered Antigens In Vivo

5A: Tregitope Co-Administration Causes Suppression of Effector Responsesto Co-Administered Protein Therapeutic In Vivo.

It is shown herein that Tregitopes suppress response to a therapeuticprotein of bacterial origin, which is referred to as “ANTIGEN-XX” (FIG.14). ANTIGEN-XX has caused significant immunogenicity in humans inunpublished studies. Whether the Tregitopes of the invention couldsuppress the effector immune response protein in vivo was investigated.HLA DR4 Transgenic mice (4-6 wk female) were injected weekly 3×subcutaneously (scruff of the neck) with either 1) 50 μg ANTIGEN-XXalone, 2) 50 μg ANTIGEN-XX+25 μg murine Tregitope-167 and 25 μg murineTregitope 106 or 3) PBS sham control. Splenocytes were harvested andplated in murine IL-4 elispot plates as described above.

Quantification of IgG antibody to ANTIGEN-XX was determined byantibody-capture ELISA as described above. ANTIGEN-XX (10 μg/mL) wasdissolved in carbonate buffer (10 mM Na₂CO₃ and 35 mM NaHCO₃ [pH 9]) andplaced into a 96-well microtiter plate overnight at 4° C. The plateswere then washed with phosphate-buffered saline containing 0.05% Tween20 (PBST) and blocked for 3 hours at room temperature with 5% fetalbovine serum (FBS; Gibco) in PBS. Serial dilutions of sera in 0.5%FBS/PBS were added to the plates and incubated at room temperature for 2hours. The microtiter plates were then washed with PBST and 100 μL goatanti-mouse IgG (gamma-chain specific) conjugated to horseradishperoxidase (Southern Biotechnology Associates) diluted 1:10000 in 0.5%FBS/PBS is added to each well. Microtiter plates are washed in PBST andthen developed with 3,3′,5,5′-tetramethylbenzidine (TMB; Moss).Absorbances were read at a wavelength of 450 nm measured on a WallacVictor3. Correction for optical imperfections in the plate is made bysubtraction of intensities at 540 nm from the 450 nm values. Response topositive control PHA was robust following both immunization conditionsand both assay readouts.

This study confirms the suppressive effects of the murine homologues ofhuman Tregitopes co-administered with antigen in vivo.

5B: Tregitope Co-Administration Causes Suppression of Effector Responsesto Co-Administered Allergen In Vivo.

Dust mites cause significant allergic responses in humans, and the mousemodel using house dust mite lysate (HDML) is accepted as a model that issimilar to humans. Whether the Tregitopes of the invention couldsuppress the effector immune response to HDML in vivo was investigated.HLA DR4 Transgenic mice (4-6 wk female) were injected weekly 3×subcutaneously (scruff of the neck) with either 1) 50 μg HDML alone, 2)50 μg HDML+50 μg murine homologue of Tregitope-289 or 3) PBS shamcontrol. In a fourth arm, mice were first presensitized to HDML through3 weekly injections of 50 μg and then treated with coinjections of HDML(50 μg) and Tregitope-289). One week following the final injections,mice were sacrificed.

Splenocytes were harvested and plated in murine IL-4 ELISpot plates asdescribed above; to the plated cells were added (in triplicate): PBS (nostimulus control), HDM Lysate, purified HDM antigen DerP2, and PHA. HDMDerP2 is a component of HDM Lysate.

Serum was obtained by cardiac puncture. Quantification of IgG antibodyto HDM antigen was determined by antibody-capture ELISA as describedabove. HDM antigen DerP2 (10 μg/mL) was placed into a 96-well microtiterplate overnight at 4° C. The plates were then washed withphosphate-buffered saline containing 0.05% Tween 20 (PBST) and blockedfor three hours at room temperature with 5% fetal bovine serum (FBS;Gibco) in PBS. Serial dilutions of sera in 0.5% FBS/PBS were added tothe plates and incubated at room temperature for two hours. Themicrotiter plates were then washed with PBST and 100 μL goat anti-mouseIgG (gamma-chain specific) conjugated to horseradish peroxidase(Southern Biotechnology Associates) diluted 1:10000 in 0.5% FBS/PBS isadded to each well. Microtiter plates are washed in PBST and thendeveloped with 3,3′,5,5′-tetramethylbenzidine (TMB; Moss). Absorbanceswere read at a wavelength of 450 nm measured on a Wallac Victor3.Correction for optical imperfections in the plate is made by subtractionof intensities at 540 nm from the 450 nm values. Response to positivecontrol PHA was robust following both immunization conditions and bothassay readouts (FIG. 15).

This study confirms the suppressive effects of the murine equivalents ofhuman Tregitopes co-administered with DM antigen in vivo.

5C: Tregitope Co-Administration Causes Suppression of Effector Responsesto Co-Administered Therapeutic In Vivo.

To test whether Tregitope co-administration in vivo would be able tosuppress immune responses to an immunogenic therapeutic protein, HLADRB1*0401 was injected into mice three times weekly with preparations of50 μg immunogenic protein therapeutic (“IPT”) alone or in combinationwith either 50 μg Tregitope-289 (murine homologue) or IPT in combinationwith the murine Fc. Co-administration of IPT with murine Fc regionreduced the IL-4 response, however, in vivo co-administration of “IPT”with murine homologue Tregitope-289, led to an even greater decrease inIL-4 by ELISpot (FIG. 16).

Example 6. Generation of a FVIII-Tregitope Construct

Fusion of Tregitope with an immunogenic protein can lead to theinduction of peripheral tolerance of the immunogenic protein. ClottingFactor VIII is immunogenic in people with severe hemophilia A. Chimericconstructs comprised of the coding sequence of Factor VIII and Tregitopeare produced (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ed., Cold Spring Harbor Laboratory Press, (1989)). Briefly, the FactorVIII coding region fused at the carboxyterminal to a Tregitope isgenerated by annealing overlapping oligos and sub-cloned into anexpression plasmid. The plasmids are transfected into DG44 CHO cells andstable transfectants selected. The chimeric protein is purified over aimmunoaffinity column and evaluated for tolergenicity. Table 1illustrates one embodiment of such a chimeric protein.

TABLE 1 Factor VIII-Tregitope (Tregitope bold)MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNTYPHGITDVRPLYSRRLPKGVKHLKDEPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPTYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPSNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYEEQYNSTYRVVSVL TVLHQDW SEQ ID NO: 1

Example 7. Generation of a FVIII-Multi-Tregitope Construct

Multiple Tregitopes can be present in highly immunogenic proteins topromote adaptive tolerance. Chimeric constructs comprised of the codingsequence of clotting Factor VIII and multiple Tregitope(s) are produced(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ed., ColdSpring Harbor Laboratory Press, (1989)). Briefly, the Factor VIII codingregion fused at the carboxyterminal to a Tregitope is generated byannealing overlapping oligos and sub-cloned into an expression plasmid.The plasmids are transfected into DG44 CHO cells and stabletransfectants selected. The chimeric protein is purified over aimmunoaffinity column and evaluated for tolergenicity. Table 2illustrates one embodiment of such a chimeric protein.

TABLE 2 Factor VIII-multiTregitope (Tregitope(s) bold)MQIELSTCFFLCLLRFCFSATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNTYPHGITDVRPLYSRRLPKGVKHLKDEPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPTYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPSNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYEEQYNSTYRVVSVLTVLHQDWEEQYNSTYRVVSVLTVLHQDWEEQYNSTYRVVSVLT VLHQDWEEQYNSTYRVVSVLTVLHQDWSEQ ID NO: 2

Example 8. Generation of an Enhanced Vaccine Delivery Vehicle

Fc binding to Fc receptors enhance uptake in antigen presenting cellspresentation to T and B lymphocytes. Tregitope-289, located in the Fcdomain of IgG molecules acts to deliver suppressive signals. Themodification of Fc so that Tregitope-289 no longer binds to MHCmolecules and regulatory T cells allows for efficient targeting ofvaccine candidates while avoiding suppressive effects. Modifications todecrease binding of Tregitopes to MHC molecules are useful. FIG. 22illustrates such a modification. Chimeric constructs comprised ofvarious proteins or epitope pseudo-proteins of interest and a Tregitopemodified mlgG Fc are designed (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2 ed., Cold Spring Harbor Laboratory Press, (1989)).Briefly, the protein or epitope pseudo-protein of interest is generatedby annealing overlapping oligos and sub-cloned into a Tregitope modifiedFc fusion expression plasmid. The plasmids are transfected into DG44 CHOcells and stable transfectants selected. The chimeric protein homodimersare purified over a protein A column and evaluated for immunogenicity.FIG. 22 illustrates one embodiment of a chimeric protein where thepseudo-protein of interest is a string of immunogenic T cell epitopesderived from the Epstein Barr Virus (EBV) fused to a modified Fc proteinin which the Tregitope has been modified to no longer bind MHC class IImolecules and can not stimulate natural regulatory T cells.EBV-Tregitope modified Fc SEQUENCE (Kb SIGNAL SEQUENCE) in FIG. 22 isdesignated as underlined text. The Tregitope is designated as bold text.The Tregitope modified amino acids are designated as shaded text. Thehuman Fc region is designated as italicized text.

REFERENCES

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EQUIVALENTS

While the invention has been described in connection with the specificembodiments thereof, it will be understood that it is capable of furthermodification. Furthermore, this application is intended to cover anyvariations, uses, or adaptations of the invention, including suchdepartures from the present disclosure as come within known or customarypractice in the art to which the invention pertains, and as fall withinthe scope of the appended claims.

1-23. (canceled)
 24. A chimeric polypeptide comprising a first and asecond polypeptide chain linked together, wherein said first polypeptidechain is a fragment of an immunoglobulin consisting of the amino acidsequence of SEQ ID NO: 12, wherein the second polypeptide chaincomprises a biologically active molecule, and wherein said biologicallyactive molecule is not an immunoglobulin polypeptide.
 25. The chimericpolypeptide of claim 24, wherein the second polypeptide chain is fusedto the N-terminus of the first polypeptide chain.
 26. The chimericpolypeptide of claim 24, wherein the second polypeptide chain is fusedto the C-terminus of the first polypeptide chain.
 27. The chimericpolypeptide of claim 24, wherein the chimeric polypeptide furthercomprises at least one isolated T-cell epitope polypeptide consisting ofan amino acid sequence selected from the group consisting of SEQ ID NOS:6 and 8-22, 24-30, and 32-58.
 28. The chimeric polypeptide of claim 24,wherein the biologically active molecule is selected from the groupconsisting of a viral protein, a bacterial protein, a FVIII molecule, anautoimmune antigen, and an allergen.
 29. The chimeric polypeptide ofclaim 24, wherein the first and the second polypeptide chain linkedtogether are operatively linked.
 30. A method of inducing regulatoryT-cells to suppress immune response in a subject comprisingadministrating to the subject a therapeutically effective amount of achimeric polypeptide, wherein the chimeric polypeptide comprises a firstand a second polypeptide chain linked together, wherein said firstpolypeptide chain is a fragment of an immunoglobulin consisting of theamino acid sequence of SEQ ID NO: 12, wherein the second polypeptidechain comprises a biologically active molecule, and wherein saidbiologically active molecule is not an immunoglobulin polypeptide. 31.The method of claim 30, wherein the second polypeptide chain is fused tothe N-terminus of the first polypeptide chain.
 32. The method of claim30, wherein the second polypeptide chain is fused to the C-terminus ofthe first polypeptide chain.
 33. The method of claim 30, wherein thechimeric polypeptide further comprises at least one isolated T-cellepitope polypeptide consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 6 and 8-58.
 34. The method of claim30, wherein the biologically active molecule is selected from the groupconsisting of a viral protein, a bacterial protein, a FVIII molecule, anautoimmune antigen, and an allergen.
 35. The chimeric polypeptide ofclaim 24, wherein the second polypeptide chain consists of abiologically active molecule, wherein said biologically active moleculeis not an immunoglobulin polypeptide.
 36. The chimeric polypeptide ofclaim 24, wherein the second polypeptide chain consists of abiologically active molecule, wherein said biologically active moleculeis selected from the group consisting of a viral protein, a bacterialprotein, a FVIII molecule, an autoimmune antigen, and an allergen. 37.The chimeric polypeptide of claim 24, wherein the chimeric polypeptideconsists of the first and the second polypeptide chain linked together,wherein the second polypeptide chain consists of a biologically activemolecule, wherein said biologically active molecule is not animmunoglobulin polypeptide.
 38. The chimeric polypeptide of claim 24,wherein the chimeric polypeptide consists of the first and the secondpolypeptide chain linked together, wherein the second polypeptide chainconsists of a biologically active molecule, and wherein saidbiologically active molecule is selected from the group consisting of aviral protein, a bacterial protein, a FVIII molecule, an autoimmuneantigen, and an allergen.
 39. The method of claim 30, wherein the secondpolypeptide chain consists of a biologically active molecule, whereinsaid biologically active molecule is not an immunoglobulin polypeptide.40. The method of claim 30, wherein the second polypeptide chainconsists of a biologically active molecule, wherein said biologicallyactive molecule is selected from the group consisting of a viralprotein, a bacterial protein, a FVIII molecule, an autoimmune antigen,and an allergen.
 41. The method of claim 30, wherein the chimericpolypeptide consists of the first and the second polypeptide chainlinked together, wherein the second polypeptide chain consists of abiologically active molecule, wherein said biologically active moleculeis not an immunoglobulin polypeptide
 42. The method of claim 30, whereinthe chimeric polypeptide consists of the first and the secondpolypeptide chain linked together, wherein the second polypeptide chainconsists of a biologically active molecule, and wherein saidbiologically active molecule is selected from the group consisting of aviral protein, a bacterial protein, a FVIII molecule, an autoimmuneantigen, and an allergen.